Process for obtaining fungal resistant plants with recombinant polynucleotides encoding β-1,3-glucanase modified for apoplast targeting

ABSTRACT

Plants are provided with improved resistance against pathogenic fungi. They are genetically transformed with one or more polynucleotides which essentially comprise one or more genes encoding plant and β-1,3-glucanases. Preferred are the intracellular forms of the said hydrolytic enzymes, especially preferred are those forms which are targeted to the apoplastic space of the plant by virtue of the modification of the genes encoding the said enzymes. Particularly preferred are plants exhibiting a relative overexpression of at least one gene encoding a β-1,3-glucanase.

This application is a continuation of application Ser. No. 07/647,831,filed Jan. 29, 1991, now abandoned.

FIELD OF THE INVENTION

The invention lies in the area of recombinant DNA technology, especiallyin conjunction with the genetic manipulation of plants and concerns aprocess for obtaining fungal resistant plants due to geneticmanipulation, as well as genetically manipulated plants and plant cellsthemselves (including subparts of the genetically manipulated plants aswell as progeny obtained by asexual or sexual propagation) andrecombinant polynucleotides (DNA or RNA) which can be used for thegenetic manipulation.

BACKGROUND OF THE INVENTION

Most agricultural and horticultural crops are under a constant threatdue to fungal attack. To protect the crops from significant losses dueto fungal disease, the crops and sometimes the soil in which the cropsare grown are periodically treated with large amounts of fungicides.These fungicides form a heavy burden on costs of crop growing, and moreimportantly on the environment and the growers. Moreover the treatmentis very labor intensive. Therefore, there is a need for less costly andsafer methods to protect plants from fungal attack which, preferably,are devoid of the need of repeated human involvement.

Induced Resistance

In plants generally several types of resistance against pathogens occur;non-host resistance, "horizontal" or partial resistance and "vertical"resistance. None of these forms of resistance is particularly wellunderstood in molecular terms. In addition to these constitutivelyexpressed forms of resistance there is a resistance mechanism that canbe induced by certain pathogenic infections as well as by a number ofbiotic and abiotic factors. This induced resistance is very broad and isdirected against various pathogens, including fungi. This is furtherillustrated below. Inoculation of the lower leaves of a hypersensitivelyreacting tobacco cultivar (Nicotiana tabacum cv Samsun NN) with tobaccomosaic virus (TMV) results in the formation of local lesions on theinoculated leaves. The non-inoculated leaves appear resistant to asecond infection with TMV after 3 days; this resistance lasts at leasttwenty days, and an optimal resistance is obtained after 7 days. Theresistance against the second infection is also directed to otherviruses, such as tobacco necrosis virus, tobacco ringspot virus (Ross &Bozarth, 1960; Ross, 1961), and fungi, such as Thielaviopsis basicola(Hecht & Bateman, 1964), Phytophthora nicotianae and Peronosporatabacina (McIntyre & Dodds, 1979; McIntyre et al., 1981).

The phenomenon of induced resistance has been studied in numerous otherhost plants and in combination with several other pathogens as well(Kuc, 1982; Sequeira, 1983). The general picture emerging from thesestudies is that a hypersensitive response is accompanied by resistanceagainst a broad range of pathogens, irrespective of the type of pathogenhaving caused the first infection.

Proteins Expressed Concomitant with Induced Resistance

Together with the resistance a great number of proteins is synthesized,which are absent before infection.

Roughly three categories of proteins can be discerned.

1) Key-enzymes in the synthesis of secondary metabolites, such asphytoalexins, which exhibit an antimicrobial effect, and precursors oflignin, which is used in the reinforcement of plant cell walls afterpathogen invasion. These enzymes, or their messenger RNAs are mainlyfound in cells in the immediate vicinity of the site of infection(Elliston et al., 1976; Cramer et al., 1985; Bell et al., 1986).

2) Hydroxyproline rich glycoproteins (HRGPs) or extensins, which can beincorporated into the cell wall and possibly function there as a matrixfor the attachment of aromatic compounds like lignin (Fry, 1986). HRGPsare important structural components of plant cell walls, and theiraccumulation occurs in reaction to fungi, bacteria and viruses (Mazau &Esquerre-Tugaye, 1986). In contrast to the situation with thekey-enzymes mentioned above, HRGPs and their mRNAs are found insubstantial amounts in non-infected parts of the plant as well as aroundthe site of infection (Showalter et al., 1985).

3) A third group of induced genes encodes proteins which accumulate bothinside the cells and in the apoplastic space. Among these proteins arehydrolytic enzymes such as chitinases and glucanases. After a necroticinfection these enzymes can often be found throughout the plant,including the non-infected parts, in higher concentrations than beforeinfection. Increased synthesis of these enzymes appears to be inducedalso by microbial elicitors, usually fungal cell wall preparations(Darvill & Albersheim, 1984; Toppan & Esquerre-Tugaye, 1984; Mauch etal., 1984; Chappel et al., 1984; Kombrink & Hahlbrock, 1986; Hedrick etal., 1988).

Structure of Fungal Cell Walls

The cell walls of fungi are known to consist of a number of differentcarbohydrate polymers. Most fungi, with the exception of the Oomycetes,contain considerable amounts of chitin. Chitin is a polymer of N-acetylglucosamine molecules which are coupled via β-1,4 linkages and, infungal cell walls, are often associated with β-1,3/β-1,6 glucan,polymers of glucose with β-1,3 and β-1,6 linkages. Fungi from the groupof Zygomycetes do not contain glucans with β-1,3 and β-1,6 linkages,while in most of the oomycetes the glucans are associated with cellulose(for an overview, vide: Wessels and Sietsma, 1981).

In Vitro Degradation of Isolated Fungal Cell Walls

It has been known for a long time that isolated cell walls of fungi canbe degraded in vitro by plant extracts (Hilborn & Farr, 1959; Wargo,1975; Young & Pegg, 1982) and also by chitinase and β-1,3-glucanasepreparations from microbial origin (Skujins et al., 1965; Hunsley &Burnett, 1970; Jones et al., 1974).

More recently a purified endo-β-1,3-glucanase from tomato in combinationwith an exo-β-1,3-glucanase of fungal origin were shown to be capable ofhydrolysing isolated cell walls of the fungus Verticillium albo-atrum.Each of the preparations separately did not have activity (Young & Pegg,1982). A purified β-1,3-glucanase from soybean (Keen & Yoshikawa, 1983),as well as a purified chitinase from bean (Boller et al., 1983) havealso been shown to be capable of degrading isolated cell walls of fungiin vitro. When pea chitinase and β-1,3-glucanase were tested on isolatedcell walls of Fusarium solani, both appeared to be active; incombination they appeared to work synergistically (Mauch et al., 1988b).

It is not known whether these hydrolytic enzymes can degrade the polymercompounds in cell walls of living fungi effectively, if at all.

Inhibition of Fungal Growth on Synthetic Media by Chitinases andGlucanases from Plant Origin

Some chitinases and glucanases of plant origin are capable of inhibitingthe growth of fungi on synthetic media. Chitinase purified from bean iscapable of inhibiting the growth of the fungus Trichoderma viride inagar plate assays (Schlumbaum et al., 1986). A combination of chitinaseand β-1,3-glucanase, both purified from pea pods, do inhibit the growthof some fungi on agar plates, whereas other fungi are not inhibited. TheAscomycete Cladosporium cucumerinum appeared slightly sensitive, whilethe oomycetes Phythophthora cactorum, Pythium aphanidermatum and Pythiumultimum are insensitive. Pea chitinase alone-has effect on the growth ofT.viride, while β-1,3-glucanase inhibits the growth of Fusarium f.sp.pisi. It was established that in these assays the inhibition of fungalgrowth was due to lysis of the hyphal tips (Mauch et al., 1988b).Apparently the hydrolytic enzymes do have access to their substrate inthe cell walls of living fungi, when grown on synthetic media, althoughat least some of the active plant hydrolytic enzymes seem to be specificto certain fungi.

Little is known about the effect of hydrolytic enzymes on fungi in thebiotrope, i.e. in the soil or on plant leaves, and although some ofthese enzymes are putative candidates for a role in fungal resistance,evidently, not all chitinases and glucanases have activity againstliving fungi.

Possibly, the stage and site of infection at which hydrolytic enzymescome into contact with the invading fungus may be of great importance.

Occurrence of Chitinases and Glucanases in Plants

As far as known, chitinases and β-1,3-glucanases occur in most if notall plant species; both in monocotyledonous and dicotyledonous plants.At least two classes of chitinases and two classes of glucanases can bediscriminated: intracellular and extracellular. Both chitinase andglucanase genes of one. particular class appears to be encoded by genefamilies.

Natural Expression of Chitinases Genes and Glucanase Genes in Plants

Chitinase and glucanase genes are known to be expressed in plants bothconstitutively and in a strictly regulated fashion.

Chitinases and β-1,3-glucanases are constitutively synthesised in rootsof tobacco plants (Felix and Meins, 1986, Shinshi et al., 1987,;Memelink et al., 1987, 1989). Nevertheless tobacco plants are notresistant to infection of Phytophthora parasitica var. nicotianae (aroot pathogen of tobacco). However, resistance against this pathogen canbe induced in tobacco plants, following inoculation with TMV (McIntyre &Dodds, 1979). This suggests that a complex of yet unknown factors otherthan, or in addition to, chitinases and glucanases, may be involved infungal resistance.

On the other hand, plant species are known which seem to be resistant tofungal infection, although no significant increase in the levels ofchitinases or glucanases can be observed. For instance, in tomato acompatible interaction with the fungus Phytophthora infestans causes asystemic resistance (Christ & Mosinger, 1989), i.e. a resistance toinfection throughout the whole plant, although chitinases or glucanasescannot be detected in such leaves (Fischer et al., 1989). Apparentlythere is no clear correlation between expression of the genes encodinghydrolytic enzymes and fungal resistance.

In addition to these observations, some chitinases exhibit a regulatedexpression pattern which does not immediately suggest a correlation withfungal resistance.

For example, genes encoding chitinases are known to be expressed in adevelopmentally regulated manner in, inter alia, tobacco flowers (Lotanet al., 1989). Glucanases are known to occur in large quantities inseedlings of barley (Swegle et al., 1989; Woodward & Fincher, 1982; Hojet al., 1988, 1989).

In tobacco cell suspensions the synthesis of intracellular chitinasesand glucanases can be inhibited by the addition of cytokinins or auxins(Mohnen et al., 1985; Felix & Meins, 1986; Shinshi et al., 1987; Bauw etal, 1987).

The synthesis of the same hydrolytic enzymes can be induced by cytokininwhen this hormone is added to the growth medium in which normal tobaccoplants are grown axenically. Under certain circumstances the planthormone ethylene can also induce the synthesis of chitinase andglucanase (Felix & Meins, 1987).

In the roots and lower leaves of both soil-grown and axenically growntobacco plants, intracellular chitinases and glucanases can be detected,while in upper leaves they can not be detected at all, or to a muchlesser extent (Felix & Meins, 1986; Shinshi et al 1987; Memelink 1987,1989). Thus, there is also organ-specific expression of theintracellular chitinases and glucanases.

The regulation of expression of the genes coding for extracellularchitinases and glucanases is hardly, or not at all, influenced bycytokinin (Memelink et al 1987., 1989). In tobacco flowers theextracellular chitinases are expressed specifically in anthers, sepalsand the ovary.

Thus, there is at least an organ-specific expression of the genes codingfor the extracellular chitinases as well.

Fungal Resistant Plants Expressing Chimeric Chitinase Genes

Notwithstanding the many still unelucidated features concerning thenature and the role of hydrolytic enzymes in fungal resistance, someinitial successes have been reported in providing plants with diminishedsensitivity to fungal attack.

In U.S. Pat. No. 4,940,840, tobacco plants expressing a bacterialchitinase gene (i.e. the chiA gene from Serratia marcescens) have beenshown to be less sensitive to the fungus Alternaria longipes.

In the International Patent Application WO 9007001 the plant speciestobacco and canola, expressing a bean chitinase under regulation of astrong viral promoter or a plant promoter, appear to be less sensitiveto two of the tested fungi, namely Botrytis cinerea and Rhizoctoniasolani. It is not known, however, whether these plants are effectivelyresistant to other fungi as well.

In European Patent Application EP-A-0 292 435 it was suggested thatresistance to certain classes of fungi may be conferred by theintroduction of a gene that expresses chitinase in the plant tissues.

Mention was made of a preference in certain cases to target geneproducts into the mitochondria, the vacuoles, into the endoplasmicvesicles or other cell parts or even into the intercellular (apoplastic)spaces.

There was no teaching of the type of chitinase or of the preferred siteof action of the chitinase, in order to obtain the desired effect.

EP-A-0 270 248 proposes a mechanism to target a bacterial gene (theβ-glucuronidase gene from E.coli) to the plant cell wall by using theleader sequence of the polygalacturonase gene from tomato. It was, interalia, proposed to target chitinases or glucanases to the plant cell wallto combat fungal attack. Results were not shown, nor was indicated whichhydrolytic enzymes should be used, or how intracellular plant proteinsmust be targeted outside the plant cell.

In the EP-A-0 332 104 genetic constructs are described comprisingchemically regulative sequences derived from plant genes, among whichthe so-called PR-genes, including those coding for chitinase andglucanase. No results of fungal resistant plants were shown.

SUMMARY OF THE STATE OF THE ART

Plants contain at least two classes of chitinases and β-1,3-glucanases:extracellular and intracellular. The expression of the genes encodingthe said hydrolytic enzymes is not constitutive, at least not in alltissues, but is among other things regulated in a developmental ortissue-specific fashion. However, the expression of the genes can alsobe induced under certain stress-conditions, such as an infection by anecrotisizing pathogen. In most cases, induction of the synthesis ofchitinases and β-1,3-glucanases is accompanied by the induction ofresistance against a broad range of pathogens, including phytopathogenicfungi. Whether there is a causal relation between fungal resistance andexpression of the genes encoding hydrolytic enzymes is not clear.

Cell walls of phytopathogenic fungi contain glucans and often a certainamount of chitin. These carbohydrate polymers are substrates forglucanases and chitinases, respectively. It is attractive to hypothesizethat both hydrolytic enzymes are responsible for the observedresistance. However, this is far from obvious, in view of manyobservations which are clearly in conflict with this hypothesis.

Hence, it is still far from clear whether hydrolytic enzymes have asignificant role in fungal resistance, or, when they appear to have so,how substantial their role in fungal resistance is. It seems at leastdoubtful that any chitinase can confer broad range protection of plantsagainst phytopathogenic fungi. Generally, it is even questionable ifchitinases and glucanases by themselves are capable of providingsufficient protection against a broad range of plant pathogenic fungi.

There is still little basic understanding of the role of hydrolyticenzymes in the complex process of acquiring (induced) fungal resistance.However, there is a need for a method to effectively protect plantsagainst (a broad range) of phytopathogenic fungi, by means of geneticmodification.

SUMMARY OF THE INVENTION

The aim of the present invention is to provide plants which haveimproved resistance to fungal attack. Thereto, plants are geneticallytransformed by introducing into the genome of the said plants at leastone recombinant DNA-construct comprising one or more genes encoding anintracellular chitinase of plant origin, under the control of a promoterwhich is not naturally associated with that gene.

More in particular the invention provides plants having improvedresistance to fungal attack, by virtue of the expression of at least onerecombinant DNA-construct that comprises a DNA-sequence, encoding atleast one intracellular plant chitinase, which is modified such that theintracellular chitinase becomes secreted into the apoplastic space.

In a preferred embodiment, the invention provides plants exhibiting amore effective protection against fungal attack due to the expression agene encoding a chitinase, preferably an intracellular chitinase, and agene encoding a glucanase, under the control of a promoter that allowssuitably strong expression, in one or more tissues.

In a still further preferred embodiment, the invention provides plantsconstitutively expressing a gene encoding an intracellular chitinase ofplant origin which is targeted to the apoplastic space and,additionally, one or more genes encoding a hydrolytic enzyme from thegroup consisting of intracellular chitinases, extracellular chitinases,intracellular glucanases and extracellular glucanases.

One especially preferred embodiment is a plant expressing the genesencoding an intracellular chitinase, an extracellular chitinase, anintracellular β-1,3-glucanase, and an extracellular β-1,3-glucanase. Ofthese, genes encoding the intracellular forms of the mentioned planthydrolytic enzymes are particularly preferred. Still more preferred isthe use of the genes encoding intracellular hydrolytic enzymes, modifiedby genetic manipulation as to provide for apoplast targeting. In orderto achieve apoplast-targeting of the intracellular hydrolytic enzymes,the 3'-end of the gene encoding the C-terminal end of the intracellularhydrolytic enzymes is modified in order to establish, upon expression ofthe genes, the absence of the C-terminal amino acids that are involvedin intracellular targeting of the respective enzymes. Generally suchmodification results in the absence of at least 3 amino acids of theC-terminal end, or as many amino acids as desired, as long as theenzymatic function and/or other relevant domains of the protein are notnegatively affected. Preferably such modification results in thedeletion of between 3 and 25 amino acids in the case of intracellularβ-1,3-glucanases, and between 3 and 10 amino acids in the case ofintracellular chitinases. More preferred are deletions of 4-8 aminoacids.

Further embodiments of the invention are the recombinant DNA molecules,comprising one or more plant expressible DNA sequences encoding at leastone intracellular chitinase of plant origin which is modified to achievetargeting of the chitinase to the intercellular space, and, if desiredadditional DNA sequences encoding one or more hydrolytic enzymesselected from the group consisting of extracellular chitinases,intracellular glucanases and extracellular glucanases.

Certain preferred embodiments are the intracellular chitinase geneslocated on the EcoRI-SstI fragment of pMOG200; the extracellularchitinase gene from petunia hybrida, located on pMOG200; theintracellular β-1,3-glucanase gene located on the XbaI-SstI fragment ofpMOG212; the gene encoding the extracellular β-1,3-glucanase which islocated on the SstI-HindIII fragment of pMOG212, or genes which areessentially homologous to the said genes.

Especially preferred are modified versions of the genes encodingintracellular forms of the said hydrolytic enzymes, which provide forapoplast-targeting. This includes the modified intracellular chitinasegene of pMOG189 (or truncated forms thereof which retain antifungalactivity), as well as modified forms of intracellular chitinase genes,which are essentially homologous to the intracellular chitinase gene ofpMOG189. Also preferred is the modified intracellular glucanase gene ofpMOG512, in which a stopcodon is introduced into the coding region toprovide for apoplast-targeting of the produced intracellularβ-1,3-glucanase.

Also claimed are cloning-, expression-, and transformation vectorscontaining DNA-sequences comprising the said genes, as well asmicroorganisms containing said DNA-sequences.

Of these vectors the plasmids pMOG200 and pMOG212, and derivates thereofare preferred.

Further embodiments of the present invention include whole fungalresistant plants obtained by the processes according to the saidinvention, protoplasts, cells, parts (such as seeds, fruits, leafs,flowers, and the like), and any other part of the plant that can bereproduced either sexually, asexually or both, and progeny of the saidplants.

The advantages and the field of application will be readily understoodfrom the following detailed description of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide sequence and the deduced amino acid sequenceof a complete cDNA corresponding to an extracellular chitinase fromPetunia hybrida. The vertical arrow shows the cleavage site of thesignal peptide (SEQ ID No.:8) and (SEQ ID No.:9).

FIG. 2 shows the nucleotide sequence and the deduced amino acid sequenceof a BamHI DNA fragment corresponding to an intracellular chitinase fromtobacco. The sequence of nucleotide 2 through 22 originates from asynthetic fragment, while the nucleotides 23-27 form the remainder ofthe EcoRI recognition site. The PstI recognition site (5'-CTGCAG-3') isfound at position 129-134. The last 21 nucleotides of the sequencesuccesively represent a filled in EcoRI recognition site, originatingfrom an EcoRI linker-molecule used for the construction of the cDNAlibrary, a SmaI and a BamHI recognition site, both originating from thepolylinker of pIC19H. The arrow shows the cleavage site of the signalpeptide (SEQ ID No.:10) and (SEQ ID No.:11).

FIG. 3 shows the nucleotide sequence and the deduced amino acid sequenceof a gene coding for an extracellular β-1,3-glucanase from tobacco. Thevertical arrow shows the location in the amino acid sequence where thesignal peptide is cleaved. The position of the intron is indicated; thesequence of the intron is only given in part (SEQ ID No.:12), (SEQ IDNo.:13) and (SEQ ID No.:14).

FIG. 4 shows the nucleotide sequence and the deduced amino acid sequenceof a gene coding for an intracellular β-1,3-glucanase from tobacco. Thevertical arrow shows the location in the amino acid sequence where thesignal peptide is cleaved (SEQ ID No.:15), (SEQ ID No.:16) and (SEQ IDNo.:17).

FIG. 5 shows a schematic representation of expression vector pMOG181.Ampr stands for the ampicilline resistance gene. A restriction enzymerecognition site between brackets shows that the concerned site is nolonger present in the plasmid.

FIG. 6 shows a schematic representation of vector pMOG183, a derivate ofpMOG181 wherein the EcoRI recognition. site is replaced by a SstI site.

FIG. 7 shows a schematic representation of vector pMOG184, a derivate ofpMOG181 wherein the HindIII recognition site is replaced by a SstI site.

FIG. 8 shows a schematic representation of vector pMOG185, a derivate ofpMOG184 wherein the EcoRI recognition site is replaced by a XbaI site.

FIG. 9 shows a schematic representation of the binary vector pMOG23 (SEQID No.:18).

FIG. 10 shows a schematic representation of the binary vector pMOG22, aderivate of pMOG23 wherein the kanamycin resistance gene (NPTII) isreplaced by a hygromycin resistance gene (HPT) (SEQ ID No.:19).

FIG. 11 shows a schematic representation of the plasmid pMOG200, aderivate of pMOG23 wherein two expression cassettes are cloned into thepolylinker, viz., one with the coding sequence for an intracellularchitinase (ChiI) and one with the coding sequence for an extracellularchitinase (ChiE). The arrow provides the direction of the transcriptionin the cassettes, beginning with the CaMV 35S promoter.

FIG. 12 shows a schematic representation of plasmid pMOG212, a derivateof pMOG22 wherein two expression cassettes are cloned into thepolylinker, viz., one with the coding sequence for an extracellularβ-1,3-glucanase (GluE) and one with the coding sequence for anintracellular β-1,3-glucanase (GluI). The arrows give the direction oftranscription beginning with CaMV 35S promoter.

DEFINITIONS

For the purpose of the present invention it is understood that anextracellular protein is a protein which after proper expression in theoriginal plant, is localised in the apoplastic space.

Consequently, an intracellular protein is a protein which after properexpression in the plant of origin, is localised intracellularly.

The apoplastic space is defined herein as the extracellular space,including the plant cell wall.

For the purpose of this invention a protein is said to be localisedintracellularly if it is localised in any compartment of the cell thatdoes not form part of the apoplastic space; these compartments includenuclei, chloroplasts, mitochondria, vacuoles, endoplasmatic reticulum,other membranous organelles, the cytoplasm, and all membranes, includingthe plasma membrane.

Genes are said to be essentially homologous if their DNA sequencescorrespond for more than 60%, unless stated otherwise.

DETAILED DESCRIPTION OF THE INVENTION

In the light of their assumed involvement in fungal resistance, it wassurprisingly found that purified extracellular chitinases from tobaccoand petunia do not have a significant antifungal effect when compared tointracellular chitinases. In an antifungal assay, equal amounts ofchitinolytic activity of purified intracellular and extracellularchitinases, rather than equal amounts of protein, were compared. Theantifungal activity of the tested extracellular forms was practicallyundetectable.

Expression of a chimeric gene encoding an extracellular chitinase in atransformed plant as such, is therefor not sufficient to provide forfungal resistance. Nevertheless, it can not be entirely excluded thatextracellular chitinases play a supportive role in fungal resistance, byincreasing the antifungal effect of other hydrolytic enzymes present.This observation has important implications for the engineering offungal resistance in plants, based on expression of chimeric genesencoding plant hydrolytic enzymes.

Comparison of the C-terminal ends of several homologous proteins(particularly of chitinases, and glucanases), which differ essentiallyin their localisation, revealed that intracellular proteins often havean extension of about 3 to 25 (in the case of intracellularβ-1,3-glucanases), or 3 to 10 (in the case of intracellular chitinases)amino acid residues compared to their extracellular analogues. It wassurprisingly found, that deletion of about 6 amino acid residues at theC-terminal portion of an intracellular tobacco chitinase results insecretion of the protein to the apoplastic space. Apparently theC-terminal extension functions as a `vacuole-targeting` signal.

We believe this is the first demonstration of apoplast targeting ofchitinases that naturally occur in the vacuole of a plant cell. Thisfinding can be suitably applied for the targeting of vacuolar proteins(e.g. proteins which are localised in the vacuole) to the apoplasticspace.

A very effective site of action of hydrolytic enzymes in the protectionof transformed plants against a range of plant pathogenic fungi isbelieved to be the apoplastic space. Hence, to obtain improved fungalresistance it is advantageous if plants are transformed with arecombinant DNA construct comprising a gene encoding a chitinase (or atruncated form thereof, which comprises the antifungal domains or parts)which exerts its action in the apoplastic space of the plant, eithernaturally or by virtue of genetic modification.

To obtain such plants, it is preferred that plants are transformed witha recombinant DNA construct comprising a gene encoding an intracellularchitinase, which is modified such that the C-terminal amino acidsinvolved in vacuolar targeting are not present (e.g. by introducing atranslational stopcodon in the coding region of the gene, or otherwise),resulting in apoplast-targeting of (most of) the intracellular chitinaseproduced in that plant.

To evaluate the possibility of targeting intracellular hydrolyticenzymes to the apoplastic space, without a significant adverse effect onthe antifungal activity, the following experiment was carried out.

Plants were transformed with DNA constructs essentially comprising thefollowing genes:

1° a gene encoding a petunia extracellular chitinase,

2° a gene encoding a tobacco intracellular chitinase,

3° a tobacco gene encoding an intracellular chitinase, modified as toobtain apoplast-targeting of the chitinase (targeting construct), or

4° the petunia extracellular chitinase gene, and the modified tobaccointracellular chitinase gene (targeting-construct).

All genes were placed under the control of the cauliflower mosaic virus35S promoter. Of each category of the transformed plants good expressorsof the chimeric genes were selected and subjected to isolation ofextracellular fluids (EF) and total protein extracts (TE) of leaves. Theantifungal effect of the different fractions from the plants 1 through 4were determined on the test fungus Fusarium solani. Neither the EF northe TE of plant 1, expressing the petunia extracellular chitinase hadany antifungal activity, as was expected from the experiments using thepurified hydrolytic enzymes. The EF of plant 2 had residual antifungaleffect (probably due to leakage from the cell of the (relativelyover-)expressed intracellular chitinase), whereas the total proteinextract showed a strong antifungal effect. Of plant 3, expressing themodified apoplast-targeted intracellular chitinase gene, both the EF andthe TE exhibited a strong antifungal effect; this, most importantly,proves that the targeted intracellular chitinase of plant 3 still hasantifungal activity. Thus, unexpectedly, the deletion of the C-terminalvacuole targeting signal does not significantly affect the antifungalactivity of the chitinase.

Plants may be even more effectively protected against fungal attack ifthey express both an intracellular chitinase and a modified(apoplast-targeted) intracellular chitinase.

Thus the invention provides plants having improved fungal resistance, aswell as methods to obtain such plants.

In a first aspect of the present invention it has been found that theintracellular forms of tobacco and Petunia chitinases are preferred overextracellular chitinases. Therefore, intracellular chitinases arepreferred which are essentially homologous to the intracellularchitinases of tobacco. Preferably this homology of intracellular plantchitinases should be larger than 50% on the protein level, morepreferably more than 60%, most preferably more than 70%.

A second aspect of the invention is the unexpected finding that thestrong antifungal effect of intracellular chitinases is retained aftermodification of the C-terminal end of the protein. Thus, to improvefungal resistance in transformed plants the most potent hydrolyticenzymes, i.e. the intracellular forms, are selected, and thesehydrolytic enzymes, or the truncated forms, which comprise the activeantifungal domains/parts, and targeted to the apoplastic space, wheretheir antifungal effect is optimal.

In a following series of experiments the combined effect of chitinasesand glucanases in total protein extracts and extracellular fluids ofleaves of transgenic plants was investigated.

Tobacco plants were transformed with a recombinant DNA constructessentially comprising:

1) a gene encoding a tobacco intracellular β-1,3-glucanase, targeted tothe apoplast by modification of the C-terminal end;

2) a gene encoding a tobacco intracellular chitinase, and the tobaccointracellular β-1,3-glucanase, both targeted to the apoplast, bymodification of the C-terminal end of the hydrolytic enzymes.

Again, transgenic tobacco plants that were good expressors of thechimeric genes were selected, and subjected to isolation ofextracellular fluid (EF) and total protein extract (TE) of leaves. Boththe EF and TE of plant 1, expressing the intracellular β-1,3-glucanasethat was targeted to the apoplast, exhibited a weak antifungal effect onthe fungus Fusarium solani. The EF and TE of plant 2, expressing boththe apoplast-targeted intracellular chitinase and the apoplast-targetedintracellular β-1,2-glucanase, exhibited a surprisingly strongantifungal effect; this effect was slightly higher than that of the EFand TE of plant 3 of the former experiment (expressing only the geneencoding the apoplast-targeted intracellular chitinase).

It can be concluded from these experiments that modification of theC-terminal end of the intracellular β-1,3-glucanase succesfully leads toapoplast-targeting of (most of) the enzyme, and that the C-terminalmodification does not adversely affects the antifungal activity of theintracellular β-1,3-glucanase. Moreover, it is shown that the antifungaleffect of the expression of both an intracellular chimeric chitinasegene and an intracellular chimeric β-1,3-glucanase gene is larger thanthe effect of the expression of each of the genes alone.

Thus, in a third aspect of the invention plants are provided, expressinga chimeric plant chitinase gene and a chimeric plant glucanase gene,both under the regulation of the CaMV 35S promoter.

In a preferred embodiment of the present invention plants are providedwhich have been transformed with one or more genes encodingintracellular forms of plant hydrolytic enzymes, in a plant expressibleform. Especially preferred are plants which express one or more genesencoding intracellular forms of plant hydrolytic enzymes, which byvirtue of modification of the C-terminal end are targeted to theapoplast. Still further preferred are plants which are transformed withat least a gene encoding an intracellular chitinase gene and anintracellular β-1,3-glucanase gene. It will be advantageous if theselatter plants express the modified forms of the hydrolytic enzymes, toachieve apoplast-targeting of the said enzymes.

Another preferred embodiment of the invention is a plant constitutivelyexpressing an intracellular chitinase, preferably targeted to theapoplast, an extracellular chitinase, an intracellular glucanase,preferably targeted to the apoplast, and an extracellular glucanase.

In principle any combination of genes encoding plant hydrolytic enzymescan be chosen, modified or unmodified, as long as suitably highexpression of these genes does not impair cell function of thetransformed plant host. In addition to genes encoding plant hydrolyticenzymes, other plant or non-plant genes (e.g. derived from bacteria,yeast, fungi, or other sources) may be used as well.

The plant genes encoding the hydrolytic enzymes may either be endogenousor exogenous to the plant that is to be transformed.

It will be readily understood, that, in addition to the chitinase andβ-1,3-glucanase genes mentioned, genes encoding hydrolytic enzymes canbe readily isolated from other plant species as well. Moreover, thegenes as meant by the present invention may be entirely synthetic.

Genes or cDNAs coding for the desired hydrolytic enzymes can forinstance be isolated from tobacco (e.g. Legrand et al., 1987; Shinshi etal., 1987), tomato (Joosten et al., 1989), a basic intracellularchitinase can be isolated from potato (Gaynor, 1988; Kombrink et al.,1988), an extracellular chitinase can be isolated from cucumber (Metraux& Boller, 1986; Metraux et al., 1986), and both intracellular chitinasesand glucanases can be isolated from bean (Broglie et al., 1986; Vogeliet al., 1988; Mauch & Staehelin, 1989). Furthermore, chitinases andβ-1,3-glucanases can be isolated from pea, using chitosan as inducingcompound (Mauch et al., 1984). Further analysis revealed the presence ofat least five hydrolases, viz. two basic β-1,3-glucanases and threebasic chitinases (Mauch et al., 1988a). Intracellular and extracellularChitinases which are serologically related to an intracellular chitinasefrom bean can be isolated from Allium porrum L. (Spanu et al., 1989).Endochitinases and glucanases can also be isolated from maize, followinginoculation of leaves with BMV (bromine mosaic virus) (Nasser et al.,1988). Chitinases which are serologically related to an intracellularendochitinase from bean (Swegle et al., 1989) can be isolated frombarley (Hordeum vulgare). Also β-1,3-glucanases, as well as otherclasses of glucanases, can be isolated from barley (Balance et al.,1976; Hoj et al., 1988, 1989). At least 4 different chitinases and 5different β-1,3-glucanases are known to exist in oat (Fink et al.,1988).

It will be understood that sources for obtaining hydrolytic enzymes forprotecting plants against fungal attack, are not limited to the listgiven above, which is only given as illustration.

cDNAs encoding plant chitinases and β-1,3-glucanases are suitablyobtained by immunological screening of a cDNA-expression library, madeon polyA⁺ -RNA, isolated from plants after induction of the synthesis ofthe hydrolytic enzymes, using an antibody against the desired hydrolyticenzyme. In order to be expressed properly the gene must be operativelylinked to a promoter.

The choice of the promoter is dependent on the desired level ofexpression and the desired way of regulation of the gene under itscontrol. This is all within ordinary skill.

Preferably strong constitutive promoters are used which functionthroughout the whole plant, with as little as possible restrictions withrespect to developmental patterns. One example of a constitutivepromoter for high level expression is the CaMV 35S promoter. Thispromoter may be flanked by so-called enhancer sequences (Mc.Guilley etal., 1987) to further enhance expression levels. Other examples ofhigh-level, light-inducible, promoters are, among others, the ribulosebisphosphate carboxylase small subunit (rbcSSU) promoter, the chlorophyla/b binding protein (Cab) promoter, and the like. Occasionally, it maybe desirable to restrict expression of the introduced chimeric genes toone or a few pre-selected tissues, for instance those that are targetsfor fungal attack, such as roots and epidermal cells, and the like. Awell known example of a tissue-specific promoter is for example theroot-specific patatin class-II promoter. Expression of chimeric genesmay be dependent on exogenous stimuli as well, like wounding, drought,temperature, and the like.

Generally the gene(s) of choice is/are contained in an expressioncassette, which comprises at least a promoter and a transcriptionterminator, which may be foreign to the gene. It is well known how suchelements should be linked in order to function properly and this can bedetermined without practising inventive skill. Occasionally eukaryotic(genomic) genes contain introns. The presence of the latter, eithernaturally or introduced by genetic modification, is not particularlyrelevant to the invention. The techniques for gene manipulation arereadily available to a person skilled in the art (vide e.g.: Maniatis etal., 1989).

In addition to genes encoding hydrolytic enzymes also genes encodingother proteins having an extra effect on pathogen resistance may beintroduced in the plant of interest, in order to improve the effect orbroaden pathogen range. Such proteins are suitably chosen from the groupconsisting of e.g. lectins, cow pea trypsin-inhibitor (CpTI), Bacillusthuringiensis toxins, and the like.

To obtain transgenic plants capable of constitutively expressing morethan one chimeric gene, a number of alternatives are available, whichare encompass by the present invention, including the following:

A. the use of one DNA fragment or plasmid with a number of modifiedgenes physically coupled to one selection marker gene.

B. Cross-pollination of transgenic plants which are already capable ofexpressing one or more chimeric genes coupled to a gene encoding aselection marker, with pollen from a transgenic plant which contains oneor more gene constructions coupled to another selection marker.Afterwards the seed, which is obtained by this crossing, is selected onthe basis of the presence of the two markers. The plants obtained fromthe selected seeds can afterwards be used for further crossing.

C. The use of a number of various DNA fragments of plasmids, each havingone or more chimeric genes and one other selection marker. If thefrequency of cotransformation is high, then selection on the basis ofonly one marker is sufficient. In other cases, the selection on thebasis of more than one marker is preferred.

D. Consecutive transformations of transgenic plants with new, additionalchimeric genes and selection marker genes.

E. Combinations of the above mentioned strategies. The actual strategyis not critical with respect to the described invention and can beeasily determined depending on factors such as the desired construct,the materials available and the preference of the skilled workers.

For the transformation of plants several techniques are available. Thechoice of the technique is generally not critical to the invention, aslong as the transforming genetic construct, comprising the genes andregulatory elements according to the invention, can be introduced into aplant and become stably integrated into the genome of that plant. Byplant is meant any dicotyledonous or monocotyledonous plant, includingprogeny, or parts of such plants, cells or protoplasts, and the like,and any other plant material that is amenable to transformation andsubsequent regeneration into a whole plant.

Some examples for purposes of illustration are transformation ofprotoplasts using the calcium/polyethylene glycol method (Krens et al.,1982; Negrutiu et al., 1987), electroporation (ref.) and microinjection(Crossway et al., 1986), (coated) particle bombardment (Klein et al.,1987), infection with viruses and the like. After selection and/orscreening for the tranformed plant material, the transformed material isregenerated into whole plants, using methods known in the art.

Subsequently transformed plants are evaluated for the presence of thedesired properties and/or the extent to which the desired properties areexpressed. A first evaluation may include the level of expression of thenewly introduced genes, the level of fungal resistance of thetransformed plants, stable heritability of the desired properties, fieldtrials and the like.

Secondly, if desirable, the transformed plants can be cross-bred withother varieties, for instance varieties of higher commercial value orvarieties in which other desired characteristics have already beenintroduced, or used for the creation of hybrid seeds, or be subject toanother round of transformation and the like.

Plants, or parts thereof of commercial interest, with improvedresistance against phytopathogenic fungi can be grown in the field or ingreenhouses, and subsequently be used for animal feed, directconsumption by humans, for prolonged storage, used in food- or otherindustrial processing, and the like. The advantages of the plants, orparts thereof, according to the invention are the decreased need forfungicide treatment, thus lowering costs of material, labor, andenvironmental pollution, or prolonged shelf-life of products (e.g.fruit, seed, and the like) of such plants.

Any plant species or variety that is subject to some form of fungalattack may be transformed with one or more genetic constructs accordingto the invention in order to decrease the rate of infectivity and/or theeffects of such attack. As a matter of illustration the species of thefollowing, non-limitative, list are of particular interest: edibleflowers, such as cauliflower (Brassica oleracea), artichoke (Cynarascolymus) (edible flowers); decorative flowers, such as Chrysanthemum,lily, Rosa; edible fruit, such as apple (e.g. Malus domesticus), banana,berries (e.g. currant, Ribes rubrum), sweet cherry (Prunus avium),cucumber (Cucumis sativus), grape (Vitis vinifera), lemon (Citruslimon), melon (Cucumis sativus), nuts (e.g. walnut Juglans regia),orange, peaches (Prunus persica), pear (Pyra communis), pepper (Solanumcapsicum), prunes (Prunus domestics), strawberry (Fragaria), tobacco(Nicotiana), tomato (e.g. LycoPersicon esculentum); leaf(y) vegetables,such as cabbages (Brassica), endive (Cichoreum endivia), lettuce(Lactuca sativa), spinach (Spinacia oleraceae), leek (Allium porrum);edible roots, such as beet (Beta vulgaris), carrot (Daucus carota),turnip/swede (Brassica rapsa), radish (Raphanus sativus) (edible roots);edible seeds, such as bean (Phaseolus), pea (Pisum sativum), soybean(Glycin max), wheat (Triticum aestivum), barley (Hordeum vulgare), corn(Zea mays), rice (Oryza); edible tubers, such as kohlrabi, potato(Solanum tuberosum), and the like.

The following enabling Examples serve to further illustrate theinvention, and are not intended to define limitations or restrict thescope of the subject invention.

EXPERIMENTAL EXAMPLE 1

Assay for Antifungal Activity

The effect of various protein solutions on fungal growth was assessed ina microtiter plate assay. In each well of a 24-well microtiter dish 250μl potato dextrose agar (PDA) was pipetted. Fungal spores were suspendedin water and 300-500 spores in 50 μl were added to the wells. Sporeswere pregerminated overnight. Subsequently 100 μl filter sterilized(0.22 μm filter) protein solutions were added. As controls proteins wereboiled for 10 minutes. Microtiter dishes were covered with Parafilm andincubated at room temperature. After 1-2 days the mycelium of thegrowing fungus in the wells was stained with lactophenol cotton blue andthe extent of growth was estimated.

EXAMPLE 2

Assay for Chitinase Activity

Chitinase activity was assayed radiometrically with tritiated chitin assubstrate (Molano et al., 1977) Tritiated chitin was synthesized byacytelation of chitosan with tritiated anhydride (Molano et al., 1977).The specific activity of the final product was approximately 1.2×10⁶cpm/mg. Before use the tritiated chitin was washed three times. To 100μl 10 mM potassium phosphate buffer pH 6.4 with 0.02% sodium azide, 50μl tritiated chitin (approximately 150,000 cpm) and 50 μl proteinsolution was added. The mixture was incubated shaking for 30 minutes at37° C. The reaction was stopped by adding 600 μl 10% trichloro aceticacid. After centrifugation to pellet the chitin (10 minutes in amicrofuge), 500 μl supernatant was filtered over glasswool and pipettedinto a scintillation vial. 5 ml scintillation fluid was added and theradioactivity was counted. The amount of radioactivity released(expressed as counts per minute) was taken as a measure for chitinaseactivity.

EXAMPLE 3

Antifungal Activity of Chitinase

Antifungal activity of chitinases was assessed by the microtiter plateassay described above using the fungus Fusarium solani. Two purifiedextracellular tobacco chitinases (also known as pathogenesis-relatedproteins P and Q), a purified intracellular tobacco chitinase (32 kdprotein) and a purified extracellular petunia chitinase were tested. Inall cases the added activity was approximately 2000 counts per minute(meaning that this activity releases 2000 cpm from tritiated chitin inthe chitinase assay). This activity is within in the range in whichthere is a linearity between protein concentration and activity. Ascontrols bovine serum albumin (BSA), buffer or heat-inactivatedchitinase was added. The results are shown in Table I.

                  TABLE I                                                         ______________________________________                                        Inhibition of the growth of Fusarium solani by                                  chitinases.                                                                     Protein added          inhibition                                         ______________________________________                                        petunia extracellular chitinase                                                                      -                                                        petunia extracellular chitinase, boiled -                                     tobacco extracellular chitinase (PR-P) -                                      tobacco PR-P, boiled -                                                        tobacco extracellular chitinase (PR-Q) -                                      tobacco PR-Q, boiled -                                                        tobacco intracellular 32 kd chitinase +                                       tobacco 32 kd intracellular chitinase, boiled -                               BSA -                                                                         buffer -                                                                    ______________________________________                                         -: no inhibition; +: inhibition                                          

From the results in Table I it can be concluded that the extracellularchitinases of tobacco and of petunia do not possess antifungal activity.

EXAMPLE 4

4.0 The Cloning of cDNAs Corresponding with Chitinase

Polyadenylated RNA was isolated from TMV-infected Samsun NN tobacco anddouble stranded cDNA was made using oligo(dT) as a primer (Hooft vanHuijsduijnen et al., 1986) using standard techniques known toresearchers in this area. The double stranded DNA was provided with"C-tails" which were hybridized with "G-tails" which were brought intothe plasmid pUC9 after this plasmid was spliced open with PstI (Maniatiset al., 1982). The constructs obtained were used for the transformationof Escherichia coli MH-1. The transformants were brought in duplo onnitrocellulose filters. The first filter was hybridized in vitro withtranscribed cDNA of poly(A)-RNA from TMV-infected tobacco, the otherfilter was hybridized with cDNA against poly(A)-RNA from healthy tobacco(Maniatis et al., 1982). Transformants which showed better hybridizationwith the first probe than with the second contained cDNA correspondingwith mRNAs whose synthesis was induced via the TMV infection. The cDNAclones obtained could be subdivided into six clusters on the basis ofcross-hybridizations of the insertions: within a cluster, the insertionsof all clones hybridize with each other, between clusters nocross-hybridizations took place (Hooft van Huijsduijnen et al., 1986)under the hybridization and wash conditions used (0.1 SSC, 1% SDS, 65°C.; Maniatis et al., 1982). The TMV-inducibility of the synthesis ofmRNAs corresponding with the insertions of the clones of the sixclusters, were confirmed via Northern blot analyses, well known toresearchers in this area (Hooft van Huijsduijnen et al., 1986).

Via immunoprecipitations of in vitro translation-products of mRNAs bymeans of selective hybridization with (the insertions of) cDNA clonesfrom the six clusters, it was established that the clones of twoclusters, namely clusters D and F, correspond with mRNAS for proteinsserologically related to the so-called PR-proteins P and Q (Hooft vanHuisduijnen et al., 1987). The experiments were conducted according tostandard techniques known to researchers in this area. The PR-proteins Pand Q were already earlier identified as extracellular acidicchitinases, and antibodies against both proteins cross-react with twobasic chitinases also present in tobacco (Legrand et al., 1987). Insertsof clones from clusters D and F were subcloned in M13-vectors and thesequence of the insertions was determined by the method of Sanger et al.(1977). One clone from cluster F, namely PROB3, appeared to contain aninsertion of 412 base-pairs, wherein an open reading frame occurs,coding for 109 amino acids wherefrom the sequence appears to beidentical to the C-terminal sequence of a basic chitinase of tobacco(Hooft van Huijsduijnen et al., 1987). The amino acid sequence of thischitinase was determined from the nucleotide-sequence of a cDNA clone,namely pCHN50 (Shinshi et al., 1987). Cluster F, including clone PROB3,consequently corresponds with one or more intracellular basic chitinasesof tobacco.

Cluster D contains one clone, namely PROB30, with an insertion of 404base-pairs, wherein an open reading frame occurs, coding for 67 aminoacids (Hooft van Huijsduijnen et al., 1987). The homology between theamino acid sequences deduced from the nucleotide-sequences of theinsertions of PROB3 and PROB30 appears to be 65%, while thenucleotide-sequences themselves showed a homology of only 56%. From thisit was concluded that PROB30 corresponds with a chitinase that isrelated to, but is not identical to the intracellular chitinase. Afterpartial amino acid sequences for PR-proteins P and Q were established,it was concluded that PROB30 corresponds with PR-protein P, anextracellular acidic chitinase of tobacco.

4.1 Construction of cDNA Clones Coding for an Entire ExtracellularChitinase

To obtain cDNA clones containing the entire coding sequence for thechitinases, clone PROB30 was used as a probe for the selection of clonesfrom a Petunia hybrida cDNA library. Double stranded cDNA wassynthesized as described above, treated with EcoRI-methylase, providedwith EcoRI-linkers, ligated to lambda gtll vector-arms and transfectedto E. coli Y1090 entirely according to the method described in theinstruction manual belonging to "cDNA cloning system-lambda gt11"(Amersham International plc, 1986). Afterwards, the newly constructedlibrary was searched with the plaque hybridization-technique of Bentonand Davis (1977) whereby the previously described acidic chitinase-cDNAclone served as a probe. In this manner, five recombinant phages wereobtained with sequences homologous to PROB30. Recombinant phage DNA wasisolated and afterwards the insertions were spliced out with EcoRI andsubcloned in a pUC plasmid, resulting in the clones, D1, D2, D5, D6 andD8. After being subcloned in sections into M13-phages, the nucleotidesequences of the original insertions were entirely or partiallydetermined. In FIG. 1, the sequence of clone D1 and a deduced amino acidsequence are provided. The first and the last 7 nucleotides originatefrom the EcoRI-linkers which were used for the construction of thelibrary. The sequence of eight A-residues at the end of the insertion,just before the EcoRI recognition site represent the remainder of thepoly(A)-tail of the original mRNA and consequently confirms theorientation of the insertion previously assigned through the large openreading frame and the homology to the deduced amino acid sequence ofother chitinases (see above). The insertion of clone D5 appears to be 10nucleotides longer on its 5' extremity than that of D1; the remainder ofthe poly(A)-tail was however, as with the insertion of D6, found 25nucleotides earlier in the sequence. For as far as could be traced, thesequences of the insertions of D8, D2 and D6 appeared to be identical tothose of D1 and D5.

The homology between the determined amino acid sequence of Petunia cloneD1 and tobacco clone PROB30 is approximately 80%. PROB30 is a partialcDNA clone which corresponds with PR-protein P, an extracellularchitinase. Analyses of transgenic plants have proven that the chitinaseencoded on D1 is extracellularly localized, at least in tobacco. D1consequently contains the entire nucleotide sequence coding for anextracellular chitinase.

In order to clone the cDNA corresponding with the extracellularchitinase on a BamHI fragment, the following experiments were performed.

Two of the oligonucleotides were synthesized, namely5'-AGCTTGGATCCGTCGACGGTCCT-3'(SEQ ID No.:1) and5'-AATTAGGATCCGTCGACGGATCCA-3'(SEQ ID No.:2) and these were hybridizedto one another, resulting in a double stranded DNA fragment with oneextremity compatible with the HindIII recognition site and one extremitycompatible with the EcoRI recognition site. Furthermore, the fragmentcontains recognition sites for BamHI, HincII and once again, BamHI. Thisfragment is cloned in pUC19, spliced open with EcoRI and HindIII,whereby the HindIII recognition site is restored but the EcoRIrecognition site is not. The new plasmid was called pUC19+. After theextremities of the EcoRI insertion of clone D1 were filled in withKlenow polymerase according to standard techniques, the fragment wascloned into the HincII site of pUC19 +.

4.2 Construction of a cDNA Clone Coding for an Entire IntracellularChitinase

Screening of a new Samsun NN library (which was constructed in the samemanner as the Petunia library described above) with the PROB3 insertionprovided a recombinant phage. The insertion of this phage was subclonedinto a plasmid as a EcoRI fragment, resulting in clone F1. Clarificationof the primary structure showed that the nucleotide sequence of theinsertion of F1 was identical to clone pCHN50, which was characterizedby Shinshi and co-workers (1987). Because the insertion of pCHN50 hasbeen characterized as a sequence corresponding with the intracellularchitinase of tobacco, it was concluded that the insertion of F1 alsocorresponds to an intracellular chitinase. The insertion of pCHN50 doesnot contain the entire coding sequence and is consequently incomplete.Although the insertion of F1 is 30 nucleotides longer on the 5'extremity than is pCHN50, the chitinase coding sequence contained in F1is also incomplete.

To obtain a fragment with a sequence which codes for an entirechitinase, the following cloning steps were performed. The insertion ofF1 was cloned as an EcoRI fragment into pIC19H (Marsh et al., 1984) suchthat the 3' extremity of the insertion properly came to the BamHI siteof the polylinker. This resulted in plasmid pIC19/F1.

Two oligonucleotides were synthesized (5'-GATCCAACATGAGGCTGTGCA-3' (SEQID No.:3) and 5'-AATTTGCACAGCCTCATGTTG-3' (SEQ ID No.:4) which form afragment after hybridization to each other. This fragment is cloned in athree-point ligation reaction in a pUC plasmid spliced open withBamHI-PstI, together with the EcoRI-PstI fragment, with the 5' extremityof the open reading frame in the insertion of pIC19/F1. This cloningresults in pUC/5'F1. The sequence of the oligonucleotides was chosensuch that the fragment coded for five amino acids, and also such that inthe eventually obtained BamHI-PstI fragment, the EcoRI recognition sitewas eliminated and the triplets for said five amino acids were in phasewith the open reading frame in F1. After digestion of pIC19H/F1 withHindIII and (partially) with PstI, the HindIII/PstI fragment was clonedwith the 3' part of the insertion into an intermediate vector-lacking aEcoRI recognition site. The EcoRI site on the extremity of the insertionwas replaced by filling in and back-ligation, techniques known toresearchers in this area. After elimination of the EcoRI recognitionsite, the HindIII-PstI Tragment was cloned into pUC/5'F1. Thethus-obtained plasmid contains on a BamHI fragment, a cDNA with anentire coding sequence for an intracellular chitinase from tobacco. InFIG. 2, the sequence of this BamHI fragment with the deduced amino acidsequence are provided. The sequence of nucleotides 2 through 22originates from the synthetic fragment, while nucleotides 23-27 form theremainder of the EcoRI recognition site. The PstI recognition site(5'-CTGCAG-3') is found at position 129-134. The last 21 nucleotides ofthis sequence successively represent a filled in EcoRI recognition sitewhich originates from an EcoRI linker-molecule used for the constructionof the cDNA library, a SmaI and a BamHI recognition site, bothoriginating from the polylinker of pIC19H.

4.3 Construction of a Gene Encoding an Intracellular Chitinase, Modifiedas to Obtain Apoplast Targeting of the Protein.

For the construction of a gene coding for an intracellular chitinase tobe targeted to the apoplast, the sequence of the intracellular chitinasegene as shown in FIG. 2 was modified. The G at position 961 was changedinto a T, hence creating a stopcodon. A second stopcodon was introducedby the replacement of the T residue at position 968 into an A. Thechange of the T residue at position 975 into a C resulted in thecreation of a SalI-site. These modifications were introduced by using anoverlapping polymerase chain reaction (PCR) technique, known to personsskilled in the art. Afterwards, the whole sequence was checked forpossible introduction of mutations as a result of the PCR technique.

EXAMPLE 5

5.0 Cloning of Genes Coding for Extra--and Intracellular Glucanases

The previously described lambda gtll tobacco cDNA library was screenedfor recombinant phages expressing PR-2, PR-N or related sequences, withantiserum, obtained from rabbits that were immunized with tobaccoPR-proteins 2 and N. The technique used was based on methods describedby Huynh et al., (1985) and may be presumed to be known by researchersin this area. The insertion of one recombinant phage identified by thismethod, was used as a probe to rescreen the library, but this time usingthe plaque hybridization technique of Benton & Davis (1977). Using thismethod, 30 recombinant phages were identified. The insertions in the DNAof these phages were spliced open with EcoRI and subcloned into a pUCplasmid. On the basis of their various restriction patterns, thethus-obtained clones were divided into a number of groups. Aftersubcloning in M13 vectors, the nucleotide sequences of a number ofclones from each group were determined, and the amino acid sequences ofthe peptides encoded thereby was deduced. These analyses, in combinationwith the comparison of the thus-obtained sequences to sequencespreviously known, indicate that for at least 5 groups of clones, eachcodes for a unique β-1,3-glucanase. Hybridization experiments with totalRNA from tobacco, whereby one of the glucanase cDNAs was used as aprobe, showed that these glucanase mRNAs were also synthesized followinginduction with salicylate or following TMV-infection (Memelink et al.,1989).

5.1 Isolation of Genes Coding for Extracellular β-1.3-Glucanases

Using one of the above described cDNA clones, a genomic library of DNAfrom the nucleus of Samsun NN tobacco partially spliced with Sau3AI(Cornelissen et al., 1987), screened on recombinant phages with genescoding for glucanases. A number of recombinant phages were obtained fromwhich four, namely gI1, gI3, gI4 and gI9 (PR-N), were furthercharacterized. Southern blot analyses resulted in restriction maps whichshowed that each of the four clones contained an unique gene. Aftersubcloning in successive pUC plasmids and M13 vectors, sequence analyseswere carried out on gI3 and gI9. The sequence of the gene on clone gI9,together with the amino acid sequence deduced therefrom, are provided inFIG. 3. Comparisons teach that this amino acid sequence is identical tothat of the tobacco extracellular β-1,3-glucanase PR36, the amino acidsequence of which was partially clarified (Van den Bulcke et al., 1989)with the understanding that the 21st amino acid on the C-terminal end, athreonine residue, appeared not to be present in PR36. The gene hereindescribed is the first isolated and characterized DNA sequence codingfor an extracellular β-1,3-glucanase.

To clone the gene in an expression vector, the following treatments werecarried out. Using the PCR technique, known to researchers in this area,a BamHI recognition site was introduced before the gene and a HindIIIrecognition site was introduced after the gene. Afterwards the sequenceis checked for possible introduction of mutations as a result of the PCRtechnique. After ligation of the gene as a BamHI-HindIII fragment intoexpression vector pMOG183 (see under 6), and following linearization bysplicing with BamHI and HindIII, an expression unit arises on aSstI-HindIII fragment with the transcription terminator of the glucanasegene itself.

5.2 Isolation of Genes Coding for Intracellular Glucanases

A clone corresponding to an intracellular glucanase (Memelink et al.,1989) is used as a probe to search the above described genomic library.Though a large number of recombinant phages with unique insertions wereobtained, it appeared after restriction analysis that only 2 uniquegenes are concerned. The DNA from one phage, gGLB50, was furthercharacterized by Southern blot analysis, subcloning and viaclarification of the primary structure of insertions of the relevantsubclones, all of which was done using techniques known to researchersin this area. The primary structure of the gene as eventually obtained,together with the amino acid sequence deduced therefrom, are provided inFIG. 4. Comparisons teach that this amino acid sequence is extremelyhomologous to the sequence of an intracellular, basic β-1,3-glucanasefrom tobacco such as deduced from the sequences of a number ofoverlapping cDNA clones by Shinshi and co-workers (1988). Though thecDNAs possibly correspond to one another with strong relation, they arenevertheless different genes. At least one of the cDNA clones containsan insertion having a sequence identical to a part of the hereindescribed gene.

To clone the gene into an expression vector, the following steps werecarried out. Using the PCR technique, known by researchers in this area,a BamHI recognition site is introduced before the gene and an SstIrecognition site is introduced after the gene. Afterwards, the sequenceis checked for possible introduction of mutations as a result of the PCRtechnique. Following ligation of the gene as a BamHI-SstI fragment inexpression vector pMOG185 (see under Example 6), after linearization ofthe vector by digestion with BamHI and SstI, an expression unit ariseson a XbaI-SstI fragment with the transcription terminator of theglucanase gene itself.

In addition to the above, using the PCR technique, a BamHI recognitionsite is introduced before the gene. Afterwards, the sequence is checkedfor possible introduction of mutations as a result of the PCR technique.Subsequently, the BamHI-XbaI fragment conataining the glucanase gene wascloned into plasmid pIC19H (Marsh et al., 1984), after linearisation ofthe plasmid by digestion with BamHI and XbaI. After linearization ofexpression vector pMOG183 (see under 6) by digestion with BamHI andHindIII, the gene was ligated into this vector as a BamHI-HindIIIfragment, resulting in a glucanase expression unit on a SstI-HindIIIfragment with the transcription terminator of the glucanase gene itself.

5.3 Construction of a Gene Encoding an Intracellular Glucanase, Modifiedas to Obtain Apoplast Targeting of the Protein.

For the construction of a gene coding for an intracellular glucanase tobe targeted to the apoplast, modifications were made in the sequence ofthe intracellular glucanase gene described under 5.2. To this end thesequence GTCTCTGGTGG (SEQ ID No.:4) (nucleotides 1883-1893 in FIG. 4)was changed into the sequence TGATATCGTTA (SEQ ID No.:6) using the PCRtechnique. This modification results in the introduction of twostopcodons with an EcoRV recognition site inbetween. Sequences werechecked for possible introduction of mutations as a result of the PCRtechnique.

EXAMPLE 6

6.0 Construction of Expression Vectors

A high constitutive expression of genes is pending upon, inter alia, thepromoter of the genes concerned. To satisfy such demands, expressionvector pMOG181 was constructed, and is depicted in FIG. 5. To this end,the expression cassette of pROK1 (Baulcombe et al., 1986) is cloned inpUC18 as a EcoRI-HindIII fragment. This cassette contains thecauliflower mosaic virus (CaMV) 35S promoter on an EcoRI-BamHIrestriction fragment and the nopaline synthase (nos) transcriptionterminator on a BamHI-HindIII fragment. The promoter fragment consistsof the sequence beginning with the -800 residue and extending to andincluding the +1 residue of the CaMV genome, whereby position +1 is thetranscription initiation site (Guilley et al., 1982). From theliterature it is known that the duplication of the sequence between -343and -90 increases the activity of the CaMV 35S promoter (Kay et al.,1987). To obtain a promoter fragment with a double so-called enhancersequence, the following cloning steps were carried out using techniquesknown to researchers in this area. First, the sequence upstream from theNcoI recognition site at position -512 was deleted and the NcoIrecognition site itself was changed into an EcoRI recognition site. Toachieve this, the expression cassette in pUC18 was spliced open withNcoI, the thus-obtained extremities were filled in with Klenowpolymerase and an EcoRI linker was ligated into the extremities. Theplasmid obtained was spliced open with EcoRI, resulting in the deletionof the EcoRI fragment, and the extremities were filled in using Klenowpolymerase. Afterwards, the filled in AccI-EcoRV promoter fragment(position -388 to -90) was cloned into the linear plasmid, whereby theligation of the filled EcoRI to the filled-in AccI recognition sitecreated a new EcoRI site. The newly obtained plasmid, pMOG181, containsthe CaMV 35S promoter with double enhancer sequences in an expressioncassette which still lies on an EcoRI-HindIII fragment.

A number of derivates were made from pMOG181. An adaptor(5'-AATTGAGCTC-3') (SEQ ID No.:7) was cloned into the EcoRI recognitionsite, such that the EcoRI site was not recovered and a SstI recognitionsite was introduced. The resulting plasmid, pMOG183 (FIG. 6), nowcontains the expression cassette of a SstI-HindIII fragment. In the samemanner, pMOG184 was developed from pMOG181 (FIG. 7) by the replacementof the HindIII site with a SstI recognition site. Replacement of theEcoRI site in pMOG184 by a XbaI site provided pMOG185 (FIG. 8).

EXAMPLE 7

Binary Vectors

In order to introduce the chimeric chitinase and β-1,3-glucanase genesinto the genome of tobacco via Agrobacterium tumefaciens, the binaryvectors pMOG23 (FIG. 9) and pMOG22 (FIG. 10) were used. Vector pMOG23 isa derivative of vector BIN19 (Bevan, 1984). In view of this last vector,the following changes were made, which are not essential for theinvention, using techniques known to researchers in this area. In thefirst place, the positions of the left border (LB) and the right border(RB), in view of the neomycine phosphotransferase gene II (NPTII gene),are exchanged for each other. Afterwards, the orientation of the NPTIIgene is turned around such that the transcription of the gene occurs inthe direction of the LB. Finally the BIN19 polylinker is replaced with apolylinker with the following restriction enzyme recognition sites:EcoRI, KpnI, SmaI, BamHI, XbaI, SacI/XhoI and HindIII.

Vector pMOG22 is a derivate of pMOG23 wherein the NPTII gene is replacedwith a hygromycine resistance gene. The gene used codes for aEscherichia coli hygromycine phosphotransferase (HPT) and is taken fromplasmid PLG90 (Van den Elzen-et al., 1985). This plasmid is a derivateof pLG86 (Gritz et al., 1983) and contains a BamHI recognition siteextending from 19 base pairs before the translation initiation codon to20 base pairs after the stop codon of the gene. Using site directedmutagenesis, a standard recombinant DNA technique known to researchersin this area, the ATG codon four nucleotides before the translationinitiation codon is changed into an ATA codon. In the same manner, theEcoRI recognition site in the coding region of the HPT gene is changedto 5' C.AATTC 3'. Afterwards, the BamHI fragment, following filling inof both BamHI extremities using Klenow polymerase (Maniatis et al.,1982), is cloned in the BamHI recognition site of the expressioncassette of pROKI (Baulcombe et al., 1986) after both BamHI extremitieswere also filled in. In this manner an expression unit was obtained withthe HPT coding sequence between the CaMV 35S promoter and the nostranscription terminator.

EXAMPLE 8

Cloning Chimeric Genes in Binary Vectors

The cDNA coding for the extracellular chitinase (described in 4.1), theintracellular chitinase cDNA (described in 4.2) and the modifiedintracellular chitinase cDNA (described in 4.3) are cloned as BamHIfragments in pMOG181. Clones are selected, using restriction enzymeanalysis, which had the coding sequences in the proper orientation afterthe promoter. Afterwards the expression cassettes isolated asEcoRI-HindIII fragments were cloned into the binary vector pMOG23,following linearisation of this plasmid this plasmid with EcoRI andHindIII. The resulting plasmids are called pMOG196, pMOG198 and pMOG189,respectively. In addition, the expression cassette with the geneencoding the intracellular chitinase modified as to target the proteinto the apoplast, is cloned into pMOG22 as well, resulting in pMOG289.

The SstI-HindIII fragment with the expression unit for the intracellularglucanase modified as to target the protein to the apoplast (describedin 5.3), is cloned into pMOG23, resulting in pMOG512.

The cDNA coding for the extracellular chitinase is cloned as a BamHIfragment in pMOG184 and the cDNA coding for the intracellular chitinaseas a BamHI fragment in pMOG183. Following both cloning steps, clones areselected using restriction enzyme analysis which placed the codingsequences in the proper orientation after the promoter. Afterwards, bothcassettes were isolated as EcoRI-SstI and SstI-HindIII fragments,respectively, and in a three point ligation, cloned in pMOG23, followinglinearization of this plasmid with EcoRI and HindIII. The plasmidobtained, pMOG200 (FIG. 11), now contains both chitinase genes on abinary plasmid physically coupled to the NPTII gene.

The SstI-HindIII fragment with the expression unit for the extracellularglucanase and the XbaI-SstI fragment with the expression unit before theintracellular glucanase are cloned in a three point ligation reactioninto the binary vector pMOG22, following linearization of this plasmidwith XbaI and HindIII. The obtained binary plasmid, pMOG212 (FIG. 12)now contains both glucanase genes physically coupled to the hygromycineresistance gene.

EXAMPLE 9

Transgenic Plants

For the transformation of tobacco, use is made of leaf-discs (Horsch etal., 1985) originating from axenically cultured plants. The cultivationwas performed with bacterium strains, derived from Agrobacteriumtumefaciens strain LBA4404 (Hoekema et al., 1983) wherein a binaryvector was crossed in by means of mobilisation with the help from theplasmid pRK2013 (Ditta et al., 1980). The thus-obtained Agrobacteriumstrains were maintained under selection pressure (20 mg/L rifampicine,100 mg/l kanamycin), and was cultured as such for co-cultivation. Theformation of transgenic shoots was established on media with 100 mg/lkanamycin in cases where derivatives of the binary vector pMOG23 wereused, and on media with 20 mg/l hygromycin if derivatives of pMOG22 wereused. The transgenic plants obtained from the shoots were analyzed forthe expression of the newly introduced genes , using the so-calledWestern blotting technique. The Western blotting technique is known toresearchers in this area. In some cases leaf-discs were taken fromtransgenic plants to insert additional genes. Kanamycin resistantleaf-discs were cocultivated with Agrobacterium strains containingpMOG22 derivatives, and hygromycin resistant leaf-discs wereco-cultivated pMOG23 derivatives. The plants capable of the constitutiveexpression of all the introduced genes were selected, and seeds wereobtained after they were fertilized via self-pollination. F1-seedlingsof these transgenic plants were used for further analysis.

Transgenic tobacco plants were obtained transformed with either pMOG196,pMOG198, pMOG189 or pMOG512, and double transformed with eitherpMOG196+pMOG289, pMOG512+pMOG289 or pMOG200+pMOG212.

EXAMPLE 10

Targeting Intracellular Chitinase to the Apoplast

To evaluate the possibility to target the intracellular chitinase to theapoplastic space, the following experiment was carried out.

Samsun NN tobacco plants were tranformed with pMOG196 to constitutivelyexpress the Petunia extracellular chitinase gene (plant line 1); withpMOG198 to constitutively express the tobacco intracellular chitinasegene (line 2); with pMOG189 to constitutively express the modifiedintracellular chitinase gene (line 3) and with pMOG196+pMOG289 toconstitutively express the extracellular chitinase gene and theintracellular chitinase gene modified to obtain targeting to theapoplastic space. The lines of transgenic plants were selected for highexpression of each chimeric gene (up to 0.5% of total soluble proteinfraction was reached). From each of the four selected lines bothextracellular fluid (isolation procedure, vide: Parent & Asselin, 1984)and total leaf protein-extracts were prepared (Kaufmann et al., 1987)and these were tested for antifungal activity on the fungus Fusariumsolani. Chitinsase activity was detected in the extracellular fluid (EF)of plant lines 1, 3, and 4, and in the total protein extract (TE) of allplant lines (1 to 4). In the antifungal assay 100 μl of EF from lines 1,3 and 4 were added, diluted to contain a chitinase activity ofapproximately 2000 cpm (see example 2). The dilutions of EF of line 2,and the non-transgenic control tobacco were the same as the dilution ofline 1. The 100 μl of the diluted TE of the four transgenic linescontained a chitinase activity of approximately 2000 cpm. The dilutionof TE of the control was equal to that of plant line 1. The results ofthe antifungal assay are given in Table 2.

                  TABLE 2                                                         ______________________________________                                        Inhibition of growth of Fusarium solani by chitinases from                      transgenic tobacco plants                                                                   Inhibition                                                    Transgenic plant                                                                              Extracellular fluid                                                                       total extract                                     ______________________________________                                        line 1 (extracell.)                                                                           -           -                                                   line 2 (intracell.) - +                                                       line 3 (mod. intracell.) + +                                                  line 4 (extracell. + mod. + +                                                 intracellular)                                                                non-transformed - -                                                         ______________________________________                                         -: no inhibition; +: inhibition                                          

Neither the EF nor the TE of line 1, expressing the Petuniaextracellular chitinase gene, shows any antifungal effect, as expectedfrom the experiments using the purified chitinases (see Example 3). Thepresence of chitinase activity in the EF of line 1 shows that thePetunia chitinase in tobacco is targeted to the apoplastic space.

Although in most experiments neither chitinase activity nor antifungalactivity could be detected in the EF of line 2, in some experimentschitinase activity was found in the EF, probably due to leakage of the(relatively over-) expressed intracellular chitinase from the cells. TheTE of line 2 showed a strong antifungal effect. Of line 3, expressingthe modified apoplast-targeted intracellular chitinase gene, both the EFand the TE exhibited a strong antifungal effect. This proves that thetargeted intracellular chitinase of line 3 still has antifungalactivity. Apparently, deletion of (part of) the C-terminalvacuole-targeting signal does not significantly affect the antifungalactivity of the intracellular chitinase.

EXAMPLE 11

Synergistic Effect of Glucanase on Antifungal Activity of Chitinase

Samsun NN tobacco plants were transformed with pMOG512 to constitutivelyexpress the modified intracellular glucanase gene(line 1); withpMOG512+pMOG289 to constitutively express the modified intracellularchitinase gene and the modified intracellular glucanase gene (line 2)and with pMOG189 to express the modified intracellular chitinase gene(line 3; see example 10). The plant lines were selected for high levelsof expression of each chimeric gene. From each of the selected linesextracellular fluid (EF) (Parent & Asselin, 1984) and total leaf proteinextracts (TE) (Kaufmann et al., 1987) were prepared. Initial dilutionswere made of EF and TE of lines 2 and 3 to contain a chitinase activityof approximately 2000 cpm (see example 2). The initial dilutions of EFand TE of line 1 were equal to those of line 3. Subsequently, dilutionseries were made of the initial dilutions and these were tested forantifungal activity. No difference was found in antifungal activitybetween dilution series of EF and of TE. Moreover the highest antifungalactivity was found in the (diluted) extracts of line 2. Apparently, theapoplast-targeted intracellular glucanase has a synergistic effect onthe antifungal activity of the apoplast-targeted intracellularchitinase.

EXAMPLE 12

Analysis of Transgenic Plants having Combined Expression of anUnmodified Intracellular Chitinase and Glucanase and an ExtracellularChitinase and Alucanase

Phytophthora nicotianae var. nicotianae (Pnn) is a fungus which belongsto the family of Oomycetes. It is a root pathogen of tobacco, interalia. The infection of this plant leads to the wilting of leaves and/orto rotting in the base of the stem (black shank). Eventually the tobaccoplant perishes from the infection.

To evaluate the fungal resistance of transgenic plants, that expressunmodified genes encoding plant hydrolytic enzymes, the followingexperiment can be performed. Ten transgenic plants constitutivelyexpressing the two unmodified chitinase and the two unmodifiedβ-1,3-glucanase genes (unmod chitinase pmog 200; unmod gluc pmog212*),ten control plants transformed with the empty vector and tennon-transgenic plants are inoculated with a suspension in water of 2×10⁵Pnn zoospores. The suspension is pipetted onto the base of the stem inthe soil in the pot wherein the plant is grown and thereafter rinsedwith water. In the time thereafter, the plants are monitored for thedevelopment of disease symptoms. After two to three days, the controlplants and the non-transgenic plants will show the first diseasesymptoms; after 3 weeks, approximately 17 of the 20 plants will showsymptoms; a few plants will be dead. Of the transgenic plants thatconstitutively express the two chitinase and β-1,3-glucanase genes, justa few plants will show a slight wilting after 3 weeks.

In an alternative experiment, leaf-discs having a diameter ofapproximately 1 cm can obtained from the leaves of transgenic plantscapable of the constitutive expression of both chitinases and bothglucanases, from control transgenic plants and from non-transgenictobacco plants. Subsequently, 10 μl of a Pnn zoospore suspension inwater (5000 zoospores per ml) is pipetted onto the (underside of the)disks, and afterwards the disks are placed in sterile water and allowedto incubate at room temperature. Three sets of five disks can be used ineach test, thus in total, ten control disks per experiment. Theexperiment can be carried a number of times with the same consistentresult. After about a day, the first signs of a beginning infection willbe observed on the control disks. After five days, they will be fullycolonized. The disks of the transgenic plants capable of expressingchitinases and glucanases will show less severe disease symptoms, evenafter five days.

Tests with leaf disks, performed precisely as described above, can alsoperformed with spores of the fungus Thanatephorus cucumeris (anamorphRhizoctonia solani Kuhn), a basidiomycete. The inoculum concentrationused can be 5000-10000 basidiospores per ml water. After ten days thedisks are checked. The disks from the non-transgenic plants and thecontrol transgenic plants will all appear to be infected, while thedisks from the transgenic plants, expressing the chimeric chitinase andβ-1,3-glucanase genes will be much less affected.

Likewise, the sensitivity of transgenic and control plants can be testedon the fungus Alternaria alternata, an Ascomycete. This fungus causes"brown-spot" in tobacco. The experiments can be performed in the manneras described by Spurr in 1973. The inoculum concentration used can be5000-10000 conidia per ml. After 10 days, the development of"brown-spot" on the inoculated leaf material is judged according to thecriteria suggested by Spurr (1973). The non-transgenic and the controlleaf-material will show light to very heavy necroses, while theleaf-material having a constitutive expression of both chitinase andboth β-1,3-glucanase genes will show no, or much less severe diseasesymptoms (light yellow lesions).

If the experiments are carried out as described above, they will show,that the constitutive expression of an extracellular chitinase and anextracellular β-1,3-glucanase and an intracellular chitinase and anintracellular β-1,3-glucanase provides a resistance against, at least areduced susceptibility or sensitivity for fungal infections.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually incorporated by reference.

Although the foregoing invention has been described-in some detail byway-of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

DEPOSIT OF MICROORGANISMS

The Escherichia coli strain DH5α, containing the plasmid pMOG23 (CBS102.90) and the Escherichia coli strain DH5α containing the plasmidpMOG22 (CBS 101.90) were deposited on Jan. 29, 1990, at the CentraalBureau voor Schimmelcultures (CBS), Baarn, the Netherlands. The genotypeof Escherichia coli strain DH5α is : F⁻, endA1, hsdR17 (r_(k) ⁻ m m_(k)⁺), suPE44, thi-1, lambda³¹ , recA1, gyrA96, relA1, φ80dlacZ M15.

The Agrobacterium tumefaciens strain LBA4404, which is a good acceptorstrain for all binary plant transformation vectors, has been previouslydeposited on Feb. 24, 1983 and is available via the Centraal Bureau voorSchimmelcultures, Baarn, the Netherlands, under number CBS 191.83.

REFERENCES

Abeles, F. B., Bosschart, R. P., Forrence, L. E. & Habig, W. H. (1971),Plant Physiol. 47, 129-134.

Ballance, G. M., Meredith, W. O. S., & Laberge, D. E. (1976), Can. J.Plant Sci. 56, 459-466.

Baulcombe, D. C., Saunders, G, R., Bevan, M. W., Mayo, M. A. & Harrison,B. D. (1986), Nature 321, 446-449.

Bauw, G., De Loose, M., Inze, D., Van Montagu, M. & Vandekerckhove, J.(1987), Proc. Natl. Acad. Sci. USA 84, 4806-4810.

Bell, J. N., Ryder, T. B., Wingate, V. P. M., Bailey, J. A. & Lamb, C.J. (1986), Mol. Cell. Biol. 6, 1615-1523.

Benton, W. D. & Davis, R. W. (1977), Science 196, 180-182.

Bevan, M. (1984), Nucl. Acids Res. 22, 8711-8721.

Boller, T., Gehri, A., Mauch, F. & Vogeli,U. (1983), Planta 157, 22-31.

Boller, T. & Metraux, J. P. (1988), Mol. Plant Pathol. 33, 11-16.

Boller, T. & Vogeli, U. (1984), Plant Physiol. 74, 442-444.

Broglie, K. E., Gaynor, J. J. & Broglie, R. M. (1986), Proc. Natl. Acad.Sci. USA 83, 6820-6824.

Broglie, K. E., Biddle, P., Cressman, R. & Broglie, R. M. (1989), PlantCell 1, 599-607.

Chappell, J., Hahlbrock, & Boller, T. (1984), Planta 161, 475-480.

Christ, U. & Mosinger, E. (1989), Physiol. Mol. Plant Pathol. 35, 53-65.

Cohen, Y. & Kuc, J. (1981), Phytopathology 71, 783-787.

Cornelissen, B. J. C., Horowitz, J., Van Kan, J. A. L., Goldberg, R. B.& Bol, J. F. (1987), Nucl. Acids Res. 15, 6799-6811.

Cramer, C. L., Bell, J. N., Ryder, T. B., Bailey, J. A., Schuch, W.,Bolwell, G. P., Robbins, M. P., Dixon, R. A. & Lamb, C. J. (1985), EMBOJ. 4, 285-289.

Cruickshank, I. A. M. & Mandryk, M. (1960), Aust. Inst. Agric. Sci. 26,369-372.

Darvill, A. G. & Albertsheim, P. (1984), Plant Physiol. 35, 243-275.

De Loose, M., Alliotte, T., Gheysen, G., Genetello, C., Gielen, J.,Soetaert, P., Van Montagu, M. & Inze, D. (1988), Gene 70, 13-23.

De Wit, P. J. G. M., Buurlage, M. B. & Hammond, K. E. (1986), Physiol.Mol. Plant Pathol. 29, 159-172.

De Wit, P. J. G. M. & Van der Meer, F. E. (1986), Physiol. Mol. PlantPathol. 28, 203-214.

Ditta, G., Stanfield, S., Corbiu, D. & Helinski, D. (1980), Proc. Acad.Natl. Sci. USA 77, 7347-7351.

Elliston, J., Kuc, J. & Williams, E. B. (1977), Phytopathol. Z. 87,289-303.

Felix, G. & Meins, F. (1985), Planta 164, 423-428.

Felix, G. & Meins, F. (1986), Planta 167, 206-211.

Felix, G. & Meins, F. (1987), Planta 172, 386-392.

Ferraris, L., Abbattista Gentile, I. & Matta, A. (1987), J. Phytopathol.118, 317-325.

Fincher, G. B., Lock, P. A., Morgan, M. M., Lingelbach, K., Wettenhall,R. E. H., Mercer, J. F. B., Brandt, A. & Thomsen, K. K. (1986), Proc.Natl. Acad. Sci. USA 83, 2081-2085.

Fink, W., Liefland, M. & Mendgen, K. (1988), Plant Physiol. 88, 270-275.

Fischer, W., Christ, U., Baumgartner, M., Erismann, K. H. & Mosinger, E.(1989), Physiol. Mol. Plant Pathol. 35, 67-83.

Fortin, M. G., Parent, J-G. & Asselin, A. (1985), Can. J. Bot. 63,932-937.

Gaynor, J. J. (1988), Nucl. Acids Res. 16, 5210.

Gessler, G. & Kuc, J. (1982), J. Exp. Bot. 33, 58-66.

Gilpatrick, J. D. & Weintraub, M. (1952), Science 115, 701-702.

Guilley, H., Dudley, R. K., Jonard, G., Balazs, E. & Richards, K. E.(1982), Cell 30, 763-773.

Gritz, L. & Davies, J. (1983), Gene 25, 179-188.

Hadwiger, L. A. & Beckman, J. A. (1980), Plant Physiol. 66, 205-211.

Hecht, E. I. & Bateman D. F. (1964), Phytopathology 54, 523-530.

Hedrick, S. A., Bell, J. N., Boller, T. & Lamb, C. J. (1988), PlantPhysiol. 86, 182-186.

Hilborn, M. T. & Farr, W. K. (1959), Phytopath. 70, 35-39.

Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J. & Schilperoort, R. A.(1983), Nature 303, 179-180.

Hoj, P. B., Hartman, D. J., Morrice, N. A., Doan, D. N. P. & Fincher, G.B. (1989), Plant Mol. Biol. 13, 31-42.

Hoj, P. B., Macauly, B. J. & Fincher, G. B. (1981), J. Inst. Brew. 87,77-80.

Hoj, P. B., Slade, A. M., Wettenhall, R. E. H. & Fincher, G. B. (1988),FEBS Lett. 230, 67-71.

Hooft van Huijsduijnen, R. A. M., Van Loon, L. C. & Bol, J. F. (1986),EMBO J. 5, 2057-2061.

Hooft van Huijsduijnen, R. A. M., Kauffmann, S., Brederode, F. Th.,Cornelissen, B. J. C., Legrand, M., Fritig, B. & Bol, J. F. (1987),Plant Mol. Biol. 9, 411-420.

Horsch, R. B., Fry, J. E., Hoffmann, N., Wallroth, M., Eichholz, D.,Rogers, S. G. & Fraley, R. T. (1985), Science 227, 1229-1231.

Hunsley, D. & Burnett, J. H. (1970), J. Gen. Microbiol. 62, 203-218.

Jamet, E. & Fritig, B. (1986), Plant Mol. Biol. 6, 69-80.

Jones, D., Gordon, A. H. & Bacon, J. S. D. (1974), Biochem. J. 140,47-55.

Joosten, M. H. A. & De Wit, P. J. G. M. (1989), Plant Physiol. 89,945-951.

Kauffmann, S., Legrand, M., Geoffroy, P. & Fritig, B. (1987), EMBO J. 6,3209-3212.

Kay, R., Chan, A., Daly, M. & McPherson, J. (1987), Science 236,1299-1302.

Keen, N. T. & Yoshikawa, M. (1983), Plant Physiol. 71, 460-465.

Kombrink, E. & Hahlbrock, K. (1986), Plant Physiol. 81, 216-221.

Kombrink, E., Schroder, M. & Hahlbrock, K. (1988), Proc. Natl. Acad.Sci. USA 85, 782-786.

Kuc, J. (1982), Wood, R. K. S. ed. pp. 157-178. Plenum Press, New York.

Leah, R., Mikkelsen, J. D., Mundy, J. & Svendsen, I. (1987), CarlsbergRes. Commun. 52, 31-37.

Laflamme, D. & Roxby, R. (1989), Plant Mol. Biol. 13, 249-250.

Legrand, M., Kauffmann, S., Geoffroy, P. & Fritig, B. (1987), Proc.Natl. Acad. Sci. USA 84, 6750-6754.

Lotan, T., Ori, N. & Fluhr, R. (1989), Plant Cell 1, 881-887.

Mandryk, M. (1963), Adam. Aust. J. Agric. Res. 14, 315-318.

Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982), Cold Spring HarborLaboratory, CSH, New York.

Marsh, J. L., Erfle, M. & Wykes, E. J. (1984), Gene 32, 481-485.

Mauch, F., Hadwiger, L. A. & Boller, T. (1984), Plant Physiol. 76,607-611.

Mauch, F., Hadwiger, L. A. & Boller, T. (1988a), Plant Physiol. 87,325-333.

Mauch, F., Mauch-Mani, B. & Boller, T. (1988b), Plant Physiol. 88,936-942.

Mauch, F. & Staehelin, L. A. (1989), Plant-Cell 1, 447-457.

Mazau, D. & Esquerre-Tugaye, M. T. (1986), Physiol. Mol. Plant Pathol.29, 147-157.

McIntyre, J. R. & Dodds, J. A. (1979), Physiol. Plant Pathol. 15,321-330.

McIntyre, J. L., Dodds, J. A. & Hare, J. D. (1981), Phytopathology 71,297-301.

Meins, F. & Ahl, P. (1989), Plant Science 61, 155-161.

Memelink, J., Hoge, J. H. C. & Schilperoort, R. A. (1987), EMBO J. 6,3579-3583.

Memelink, J., Linthorst, H. J. M., Schilperoort, R. A. & Hoge, J. H. C.(1989), Plant Mol. Biol. ter perse.

Metraux, J. P. & Boller, T. (1986), Physiol. Mol. Plant Pathol. 28,161-169.

Metraux, J.P., Streit, L. & Staub, T. (1988), Physiol. Mol. PlantPathol. 33, 1-9.

Metraux, J. P., Burkhart, W., Moyer, M., Dincher, S., Middlesteadt, W.,Williams, S., Payne, G., Carnes, M. & Ryals, J. (1989), Proc. Natl.Acad. Sci. USA 86, 896-900.

Mohnen, D., Shinshi, H., Felix, G. & Meins, F. (1985), EMBO J. 4,1631-1635.

Molano, J., Duran, A. and Cabib, E. (1977) Chitin. Anal. Biochem. 83,648-656.

Moore, A. E. & Stone, B. A. (1972), Virology 50, 791-798.

Nasser, W., De Tapia, M., Kauffmann, S., Montasser-Kouhsari., S. &Burkard, G. (1988), Plant Mol. Biol. 11, 529-538.

Parent, J. G. & Asselin, A. (1984), Can. J. Bot. 62, 564-569.

Pegg, G. F. & Young, D. H. (1981), Physiol. Plant Pathol. 19, 371-382.

Pegg, G. F. & Young, D. H. (1982), Physiol. Plant Pathol. 21, 89-409.

Ross, A. F. (1961), Virology 14, 340-358.

Ross, A. F. & Bozarth, R. F. (1960), Phytopathology 50, 652.

Sanger, F., Nicklen, S. & Coulson, A. R. (1977), Proc. Natl. Acad. Sci.USA 74, 5463-5467.

Schlumbaum, A., Mauch, F., Vogeli, U. & Boller, T. (1986), Nature 324,365-367.

Sequeira, L. (1983), Ann. Rev. Microbiol. 37, 51-79.

Shinshi, H., Mohnen, D. & Meins, F. (1987), Proc. Natl. Acad. Sci. USA84, 89-93.

Shinshi, H., Wenzler, H., Neuhaus, J-M., Felix, G., Hofsteenge, J. &Meins, F. (1988), Proc. Natl. Acad. Sci. USA 85, 5541-5545.

Showalter, A. M., Bell, J. N., Cramer, C. L., Bailey, J. A., Varner, J.E. & Lamb, C. J. (1985), Proc. Natl. Acad. Sci. USA 82, 6551-6555.

Skujins, J. J., Potgieter, H. J. & Alexender, M. (1965), Arch. Biochem.Biophys. 111, 358-364.

Spanu, P., Boller, T., Ludwig, A., Wiemken, A., Faccio, A. &Bonfante-Fasolo, P. (1989), Planta 177, 447-455.

Stuart, I. M., Loi, L. & Fincher, G. B. (1986), Plant Physiol. 80,310-314.

Swegle, M., Huang, J-K., Lee, G. & Muthukrihnan, S. (1989), Plant Mol.Biol. 12, 403-412.

Toppan, A. & Esquerre-Tugaye, M. T. (1984), Plant Physiol. 75,1133-1138.

Van den Bulcke, M., Bauw, G., Castresana, C., Van Montegu, M. &Vandekerckhove, J. (1989), Proc. Natl. Acad. Sci. USA 86, 2673-2677.

Van den Elzen, P. J. M., Townsend, J., Lee, K. Y. & Bedbrook, J. R.(1985), Plant Mol. Biol. 5, 299-302.

Van Loon, L. C. (1975), Virology 67, 566-575.

Van Loon, L. C. & Antoniw, J. F. (1982), Neth. J. Plant Pathol. 88,237-256.

Vogeli, U., Meins,F. & Boller, T. (1988), Planta 174, 364-372.

Vogeli-Lange, R., Hansen-Gehri, A., Boller, T. & Meins, F. (1988), PlantScience 54, 171-176.

Wargo, P. M. (1975), Physiol. Plant Pathol. 5, 99-105.

Wessels, J. G. H. & Sietsma, J. H. (1981), Encyclopedia of PlantPhysiology, N. S., vol. 13B: Plant Carbohydrates II. pp. 352-394,Tanner, W., Loewus, F. A., eds. Springer, Berlin, Heidelberg, New York.

Woodward, J. R. & Fincher, G. B. (1982), Eur. J. Biochem. 121, 663-669.

Woodward, J. R., Morgan, F. J. & Fincher, G. B. (1982), FEBS Lett. 138,198-200.

Young, D. H. & Pegg, G. P. (1981), Physiol. Plant Pathol. 21, 391-417.

Young, D. H. & Pegg, G. P. (1982), Physiol. Plant Pathol. 21, 411-423.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 17                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AGCTTGGATC CGTCGACGGT CCT           - #                  - #                    23                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - AATTAGGATC CGTCGACGGA TCCA          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GATCCAACAT GAGGCTGTGC A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - AATTTGCACA GCCTCATGTT G           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GTCTCTGGTG G               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - TGATATCGTT A               - #                  - #                      - #       11                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - AATTGAGCTC                - #                  - #                      - #        10                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 966 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 25..786                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - GAATTCCTAA TAATCGCGAA AAAA ATG AAG TTC TGG GGA T - #CA GTA TTG GCA            51                                                                                         - #         Met Lys Phe Trp Gly - #Ser Val Leu Ala                            - #           1       - #        5                           - - TTG TCT TTT GTT GTG TTC TTG TTC CTA ACA GG - #A ACA CTG GCA CAA AAT           99                                                                       Leu Ser Phe Val Val Phe Leu Phe Leu Thr Gl - #y Thr Leu Ala Gln Asn            10                 - # 15                 - # 20                 - # 25       - - GTT GGT TCT ATT GTG ACA AGC GAC TTA TTT GA - #C CAG ATG CTT AAA AAT          147                                                                       Val Gly Ser Ile Val Thr Ser Asp Leu Phe As - #p Gln Met Leu Lys Asn                            30 - #                 35 - #                 40              - - AGG AAT GAT GCT AGA TGT TTT GCC GTA CGG TT - #T TAC ACT TAC GAT GCC          195                                                                       Arg Asn Asp Ala Arg Cys Phe Ala Val Arg Ph - #e Tyr Thr Tyr Asp Ala                        45     - #             50     - #             55                  - - TTC ATA GCT GCT GCC AAT TCG TTC CCA GGT TT - #T GGA ACT ACT GGT GAT          243                                                                       Phe Ile Ala Ala Ala Asn Ser Phe Pro Gly Ph - #e Gly Thr Thr Gly Asp                    60         - #         65         - #         70                      - - GAT ACT GCC CGT AAG AAA GAA ATT GCT GCC TT - #T TTC GGT CAA ACT TCT          291                                                                       Asp Thr Ala Arg Lys Lys Glu Ile Ala Ala Ph - #e Phe Gly Gln Thr Ser                75             - #     80             - #     85                          - - CAT GAA ACT ACT GGT GGT ACC TTA AGT CCA GA - #T GGT CCA TAT GCA GGA          339                                                                       His Glu Thr Thr Gly Gly Thr Leu Ser Pro As - #p Gly Pro Tyr Ala Gly            90                 - # 95                 - #100                 - #105       - - GGA TAT TGC TTT CTT AGA GAA GGC AAT CAA AT - #G GGA AAC GGA TAC TAT          387                                                                       Gly Tyr Cys Phe Leu Arg Glu Gly Asn Gln Me - #t Gly Asn Gly Tyr Tyr                           110  - #               115  - #               120              - - GGC AGA GGA CCC ATC CAA TTG ACA GGC CAA TC - #T AAC TAT GAC TTA GCT          435                                                                       Gly Arg Gly Pro Ile Gln Leu Thr Gly Gln Se - #r Asn Tyr Asp Leu Ala                       125      - #           130      - #           135                  - - GGG AAA GCT ATT GAA CAA GAC TTA GTT AAC AA - #C CCT GAT TTA GTA GCA          483                                                                       Gly Lys Ala Ile Glu Gln Asp Leu Val Asn As - #n Pro Asp Leu Val Ala                   140          - #       145          - #       150                      - - ACA GAT GCT ACT GTA TCA TTC AAA ACA GCA AT - #A TGG TTC TGG ATG ACA          531                                                                       Thr Asp Ala Thr Val Ser Phe Lys Thr Ala Il - #e Trp Phe Trp Met Thr               155              - #   160              - #   165                          - - CCA CAG GGT AAC AAG CCA TCT TGC CAC GAC GT - #T ATC ACC GGC CGA TGG          579                                                                       Pro Gln Gly Asn Lys Pro Ser Cys His Asp Va - #l Ile Thr Gly Arg Trp           170                 1 - #75                 1 - #80                 1 -      #85                                                                              - - ACG CCA TCA GCC GCC GAT ACA TCG GCG AAT CG - #T GTA CCA GGT TAT        GGT      627                                                                    Thr Pro Ser Ala Ala Asp Thr Ser Ala Asn Ar - #g Val Pro Gly Tyr Gly                          190  - #               195  - #               200              - - GTC ATT ACT AAC ATA ATT AAT GGT GGA ATT GA - #A TGT GGC AAA GGT CAG          675                                                                       Val Ile Thr Asn Ile Ile Asn Gly Gly Ile Gl - #u Cys Gly Lys Gly Gln                       205      - #           210      - #           215                  - - AAT GCA CGA GTG GAA GAT CGA ATT GGA TAT TA - #C AGG AGG AAT GTA AGT          723                                                                       Asn Ala Arg Val Glu Asp Arg Ile Gly Tyr Ty - #r Arg Arg Asn Val Ser                   220          - #       225          - #       230                      - - ATA ATG AAC GTG GCC CCT GGA GAC AAT TTG GA - #T TGT TAC AAC CAA AGG          771                                                                       Ile Met Asn Val Ala Pro Gly Asp Asn Leu As - #p Cys Tyr Asn Gln Arg               235              - #   240              - #   245                          - - AAC TTT GCC GAA GTC TAGGCTGGTC ACATTATGAG TGCAAATGT - #T ATGTAGTCAT          826                                                                       Asn Phe Ala Glu Val                                                           250                                                                            - - GGAGATGACA GTATACAACT TATATTTGAA TGTAATAAAT AAGGGATTCT CT -             #ATGCCCAT    886                                                                 - - TTATGATAGA GTGAAATATA TTATTGTTTG TCTTCTTGGA AAGAAGTAGA AC -            #CAACAGTT    946                                                                 - - CCTTTAAAAA AAAGGAATTC            - #                  - #                      - #966                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 254 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Met Lys Phe Trp Gly Ser Val Leu Ala Leu Se - #r Phe Val Val Phe Leu        1               5 - #                 10 - #                 15              - - Phe Leu Thr Gly Thr Leu Ala Gln Asn Val Gl - #y Ser Ile Val Thr Ser                   20     - #             25     - #             30                  - - Asp Leu Phe Asp Gln Met Leu Lys Asn Arg As - #n Asp Ala Arg Cys Phe               35         - #         40         - #         45                      - - Ala Val Arg Phe Tyr Thr Tyr Asp Ala Phe Il - #e Ala Ala Ala Asn Ser           50             - #     55             - #     60                          - - Phe Pro Gly Phe Gly Thr Thr Gly Asp Asp Th - #r Ala Arg Lys Lys Glu       65                 - # 70                 - # 75                 - # 80       - - Ile Ala Ala Phe Phe Gly Gln Thr Ser His Gl - #u Thr Thr Gly Gly Thr                       85 - #                 90 - #                 95              - - Leu Ser Pro Asp Gly Pro Tyr Ala Gly Gly Ty - #r Cys Phe Leu Arg Glu                  100      - #           105      - #           110                  - - Gly Asn Gln Met Gly Asn Gly Tyr Tyr Gly Ar - #g Gly Pro Ile Gln Leu              115          - #       120          - #       125                      - - Thr Gly Gln Ser Asn Tyr Asp Leu Ala Gly Ly - #s Ala Ile Glu Gln Asp          130              - #   135              - #   140                          - - Leu Val Asn Asn Pro Asp Leu Val Ala Thr As - #p Ala Thr Val Ser Phe      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Lys Thr Ala Ile Trp Phe Trp Met Thr Pro Gl - #n Gly Asn Lys Pro        Ser                                                                                             165  - #               170  - #               175             - - Cys His Asp Val Ile Thr Gly Arg Trp Thr Pr - #o Ser Ala Ala Asp Thr                  180      - #           185      - #           190                  - - Ser Ala Asn Arg Val Pro Gly Tyr Gly Val Il - #e Thr Asn Ile Ile Asn              195          - #       200          - #       205                      - - Gly Gly Ile Glu Cys Gly Lys Gly Gln Asn Al - #a Arg Val Glu Asp Arg          210              - #   215              - #   220                          - - Ile Gly Tyr Tyr Arg Arg Asn Val Ser Ile Me - #t Asn Val Ala Pro Gly      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Asp Asn Leu Asp Cys Tyr Asn Gln Arg Asn Ph - #e Ala Glu Val                             245  - #               250                                     - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1152 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: 10..981                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - GGATCCAAC ATG AGG CTG TGC AAA TTC ACA GCT CTT - # TCT TCT CTA CTC             48                                                                                 Met Arg Leu Cys Lys - #Phe Thr Ala Leu Ser Ser Leu Leu                          1      - #         5         - #         10                        - - TTT TCT CTC CTA CTC CTC TCT GCC TCG GCA GA - #A CAA TGT GGT TCG CAG           96                                                                       Phe Ser Leu Leu Leu Leu Ser Ala Ser Ala Gl - #u Gln Cys Gly Ser Gln                15             - #     20             - #     25                          - - GCG GGA GGT GCG CGT TGT GCC TCG GGT CTC TG - #C TGC AGC AAA TTT GGT          144                                                                       Ala Gly Gly Ala Arg Cys Ala Ser Gly Leu Cy - #s Cys Ser Lys Phe Gly            30                 - # 35                 - # 40                 - # 45       - - TGG TGT GGT AAC ACC AAT GAC TAT TGT GGC CC - #T GGC AAT TGC CAG AGC          192                                                                       Trp Cys Gly Asn Thr Asn Asp Tyr Cys Gly Pr - #o Gly Asn Cys Gln Ser                            50 - #                 55 - #                 60              - - CAG TGC CCT GGT GGT CCC ACA CCA CCC GGT GG - #T GGG GAT CTC GGC AGT          240                                                                       Gln Cys Pro Gly Gly Pro Thr Pro Pro Gly Gl - #y Gly Asp Leu Gly Ser                        65     - #             70     - #             75                  - - ATC ATC TCA AGT TCC ATG TTT GAT CAG ATG CT - #T AAG CAT CGC AAC GAT          288                                                                       Ile Ile Ser Ser Ser Met Phe Asp Gln Met Le - #u Lys His Arg Asn Asp                    80         - #         85         - #         90                      - - AAT GCA TGC CAA GGA AAG GGA TTC TAC AGT TA - #C AAT GCC TTT ATC AAT          336                                                                       Asn Ala Cys Gln Gly Lys Gly Phe Tyr Ser Ty - #r Asn Ala Phe Ile Asn                95             - #    100             - #    105                          - - GCT GCT AGG TCT TTT CCT GGC TTT GGT ACT AG - #T GGT GAT ACC ACT GCC          384                                                                       Ala Ala Arg Ser Phe Pro Gly Phe Gly Thr Se - #r Gly Asp Thr Thr Ala           110                 1 - #15                 1 - #20                 1 -      #25                                                                              - - CGT AAA AGA GAA ATC GCG GCT TTC TTC GCC CA - #A ACC TCC CAT GAA        ACT      432                                                                    Arg Lys Arg Glu Ile Ala Ala Phe Phe Ala Gl - #n Thr Ser His Glu Thr                          130  - #               135  - #               140              - - ACA GGA GGA TGG GCA ACA GCA CCA GAT GGT CC - #A TAC GCG TGG GGT TAC          480                                                                       Thr Gly Gly Trp Ala Thr Ala Pro Asp Gly Pr - #o Tyr Ala Trp Gly Tyr                       145      - #           150      - #           155                  - - TGC TGG CTT AGA GAA CAA TGT AGC CCC GGC GA - #C TAC TGT ACA CCA AGT          528                                                                       Cys Trp Leu Arg Glu Gln Cys Ser Pro Gly As - #p Tyr Cys Thr Pro Ser                   160          - #       165          - #       170                      - - GGT CAG TGG CCT TGT GCT CCT GGT CGG AAA TA - #T TTC GGA CGA GGC CCC          576                                                                       Gly Gln Trp Pro Cys Ala Pro Gly Arg Lys Ty - #r Phe Gly Arg Gly Pro               175              - #   180              - #   185                          - - ATC CAA ATT TCA CAC AAC TAC AAC TAC GGA CC - #T TGT GGA AGA GCC ATA          624                                                                       Ile Gln Ile Ser His Asn Tyr Asn Tyr Gly Pr - #o Cys Gly Arg Ala Ile           190                 1 - #95                 2 - #00                 2 -      #05                                                                              - - GGA GTG GAC CTC CTA AAC AAT CCT GAT TTA GT - #G GCC ACA GAT CCA        GTA      672                                                                    Gly Val Asp Leu Leu Asn Asn Pro Asp Leu Va - #l Ala Thr Asp Pro Val                          210  - #               215  - #               220              - - ATC TCA TTC AAG TCA GCT CTC TGG TTT TGG AT - #G ACT CCT CAA TCA CCA          720                                                                       Ile Ser Phe Lys Ser Ala Leu Trp Phe Trp Me - #t Thr Pro Gln Ser Pro                       225      - #           230      - #           235                  - - AAA CCT TCT TGC CAC GAT GTC ATC ATT GGA AG - #A TGG CAA CCA TCG TCT          768                                                                       Lys Pro Ser Cys His Asp Val Ile Ile Gly Ar - #g Trp Gln Pro Ser Ser                   240          - #       245          - #       250                      - - GCT GAC CGC GCA GCC AAT CGT CTC CCT GGA TT - #T GGT GTC ATC ACG AAC          816                                                                       Ala Asp Arg Ala Ala Asn Arg Leu Pro Gly Ph - #e Gly Val Ile Thr Asn               255              - #   260              - #   265                          - - ATC ATC AAT GGT GGC TTG GAA TGT GGT CGT GG - #C ACT GAC TCA AGG GTC          864                                                                       Ile Ile Asn Gly Gly Leu Glu Cys Gly Arg Gl - #y Thr Asp Ser Arg Val           270                 2 - #75                 2 - #80                 2 -      #85                                                                              - - CAG GAT CGC ATT GGG TTT TAC AGG AGG TAT TG - #C AGT ATT CTT GGT        GTT      912                                                                    Gln Asp Arg Ile Gly Phe Tyr Arg Arg Tyr Cy - #s Ser Ile Leu Gly Val                          290  - #               295  - #               300              - - AGT CCT GGT GAC AAT CTT GAT TGC GGA AAC CA - #G AGG TCT TTT GGA AAC          960                                                                       Ser Pro Gly Asp Asn Leu Asp Cys Gly Asn Gl - #n Arg Ser Phe Gly Asn                       305      - #           310      - #           315                  - - GGA CTT TTA GTC GAT ACT ATG TAATTTTATG GTCTGTTTT - #G TTGAATCCCT            1011                                                                       Gly Leu Leu Val Asp Thr Met                                                           320                                                                    - - TTGCGACGCA GGGACCAGGG GCTATGAATA AAGTTAATGT GTGAATTGTG TG -             #ATTGTCAT   1071                                                                 - - CTATGGGATC GCGACTATAA TCGTTTATAA TAAACAAAGA CTTGTCCACA AA -            #AAAAAAAA   1131                                                                 - - GGAATTAATT CCCGGGGATC C           - #                  - #                    1152                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 324 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Met Arg Leu Cys Lys Phe Thr Ala Leu Ser Se - #r Leu Leu Phe Ser Leu        1               5 - #                 10 - #                 15              - - Leu Leu Leu Ser Ala Ser Ala Glu Gln Cys Gl - #y Ser Gln Ala Gly Gly                   20     - #             25     - #             30                  - - Ala Arg Cys Ala Ser Gly Leu Cys Cys Ser Ly - #s Phe Gly Trp Cys Gly               35         - #         40         - #         45                      - - Asn Thr Asn Asp Tyr Cys Gly Pro Gly Asn Cy - #s Gln Ser Gln Cys Pro           50             - #     55             - #     60                          - - Gly Gly Pro Thr Pro Pro Gly Gly Gly Asp Le - #u Gly Ser Ile Ile Ser       65                 - # 70                 - # 75                 - # 80       - - Ser Ser Met Phe Asp Gln Met Leu Lys His Ar - #g Asn Asp Asn Ala Cys                       85 - #                 90 - #                 95              - - Gln Gly Lys Gly Phe Tyr Ser Tyr Asn Ala Ph - #e Ile Asn Ala Ala Arg                  100      - #           105      - #           110                  - - Ser Phe Pro Gly Phe Gly Thr Ser Gly Asp Th - #r Thr Ala Arg Lys Arg              115          - #       120          - #       125                      - - Glu Ile Ala Ala Phe Phe Ala Gln Thr Ser Hi - #s Glu Thr Thr Gly Gly          130              - #   135              - #   140                          - - Trp Ala Thr Ala Pro Asp Gly Pro Tyr Ala Tr - #p Gly Tyr Cys Trp Leu      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Arg Glu Gln Cys Ser Pro Gly Asp Tyr Cys Th - #r Pro Ser Gly Gln        Trp                                                                                             165  - #               170  - #               175             - - Pro Cys Ala Pro Gly Arg Lys Tyr Phe Gly Ar - #g Gly Pro Ile Gln Ile                  180      - #           185      - #           190                  - - Ser His Asn Tyr Asn Tyr Gly Pro Cys Gly Ar - #g Ala Ile Gly Val Asp              195          - #       200          - #       205                      - - Leu Leu Asn Asn Pro Asp Leu Val Ala Thr As - #p Pro Val Ile Ser Phe          210              - #   215              - #   220                          - - Lys Ser Ala Leu Trp Phe Trp Met Thr Pro Gl - #n Ser Pro Lys Pro Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys His Asp Val Ile Ile Gly Arg Trp Gln Pr - #o Ser Ser Ala Asp        Arg                                                                                             245  - #               250  - #               255             - - Ala Ala Asn Arg Leu Pro Gly Phe Gly Val Il - #e Thr Asn Ile Ile Asn                  260      - #           265      - #           270                  - - Gly Gly Leu Glu Cys Gly Arg Gly Thr Asp Se - #r Arg Val Gln Asp Arg              275          - #       280          - #       285                      - - Ile Gly Phe Tyr Arg Arg Tyr Cys Ser Ile Le - #u Gly Val Ser Pro Gly          290              - #   295              - #   300                          - - Asp Asn Leu Asp Cys Gly Asn Gln Arg Ser Ph - #e Gly Asn Gly Leu Leu      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Val Asp Thr Met                                                           - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2020 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: join(94..175 - #, 517..1463)                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - CTTCTGCTTG TCTATATAAG AAGCAGCCTA ATGGTTCCTT AAACACACAA TT -            #TCAGCTCA     60                                                                 - - AGTGTTTCTT ACTCTCTCAT TTCCATTTTA GCT ATG ACT TTA TG - #C ATT AAA       AAT     114                                                                                       - #                  - # Met Thr Leu Cys Ile Lys Asn                         - #                  - #   1               - #5              - - GGC TTT CTT GCA GCT GCC CTT GTA CTT GTT GG - #G CTG TTA ATT TGC AGT          162                                                                       Gly Phe Leu Ala Ala Ala Leu Val Leu Val Gl - #y Leu Leu Ile Cys Ser                    10         - #         15         - #         20                      - - ATC CAA ATG ATA  G GTCTCTCTCT CTCACACACA CACACTT - #TCT CTCATGATAC           215                                                                       Ile Gln Met Ile                                                                    25                                                                        - - ATGTACATGC ACCTTGTATG ATGCGGATCA ACTTATGTAC ACTAATAGCG TA -             #AATAATTT    275                                                                 - - TTACAATATA TATTAGGATT AATATATTTT AACATGTTGT GTCAGGTAAT CT -            #ACCTTATT    335                                                                 - - TATTAAGTCA CTTATTATGA ATAGTTACTT ATAGTTACTT CTGGGTGACC CG -            #ACACTATA    395                                                                 - - ATGTTGGCTA GAGAAGAACT TAAATAGAGA ATCATGGTTA GTGAGAATAT TC -            #ATTTATTC    455                                                                 - - GACACCAACT TATTTGGGGA CTGAAACTTC TTTGTAATAT ACTCTTTTTC TT -            #ACAATCCA    515                                                                 - - G  GG GCA CAA TCT ATT GGA GTA TGC TAT G - #GA AAA CAT GCA AAC AAT            56 - #0                                                                    Gly Ala Gln Ser Ile Gly Val Cys Tyr G - #ly Lys His Ala Asn Asn                        30       - #           35       - #           40                    - - TTA CCA TCA GAC CAA GAT GTT ATA AAC CTA TA - #C AAT GCT AAT GGC ATC          608                                                                       Leu Pro Ser Asp Gln Asp Val Ile Asn Leu Ty - #r Asn Ala Asn Gly Ile                    45         - #         50         - #         55                      - - AGA AAG ATG AGA ATC TAC AAT CCA GAT ACA AA - #T GTC TTC AAC GCT CTC          656                                                                       Arg Lys Met Arg Ile Tyr Asn Pro Asp Thr As - #n Val Phe Asn Ala Leu                60             - #     65             - #     70                          - - AGA GGA AGT AAC ATT GAG ATC ATT CTC GAC GT - #C CCA CTT CAA GAT CTT          704                                                                       Arg Gly Ser Asn Ile Glu Ile Ile Leu Asp Va - #l Pro Leu Gln Asp Leu            75                 - # 80                 - # 85                 - # 90       - - CAA TCC CTA ACT GAT CCT TCA AGA GCC AAT GG - #A TGG GTC CAA GAT AAC          752                                                                       Gln Ser Leu Thr Asp Pro Ser Arg Ala Asn Gl - #y Trp Val Gln Asp Asn                            95 - #                100 - #                105              - - ATA ATA AAT CAT TTC CCA GAT GTT AAA TTT AA - #A TAT ATA GCT GTT GGA          800                                                                       Ile Ile Asn His Phe Pro Asp Val Lys Phe Ly - #s Tyr Ile Ala Val Gly                       110      - #           115      - #           120                  - - AAT GAA GTC TCT CCC GGA AAT AAT GGT CAA TA - #T GCA CCA TTT GTT GCT          848                                                                       Asn Glu Val Ser Pro Gly Asn Asn Gly Gln Ty - #r Ala Pro Phe Val Ala                   125          - #       130          - #       135                      - - CCT GCC ATG CAA AAT GTA TAT AAT GCA TTA GC - #A GCA GCA GGG TTG CAA          896                                                                       Pro Ala Met Gln Asn Val Tyr Asn Ala Leu Al - #a Ala Ala Gly Leu Gln               140              - #   145              - #   150                          - - GAT CAA ATC AAG GTC TCA ACT GCA ACA TAT TC - #A GGG ATC TTA GCG AAT          944                                                                       Asp Gln Ile Lys Val Ser Thr Ala Thr Tyr Se - #r Gly Ile Leu Ala Asn           155                 1 - #60                 1 - #65                 1 -      #70                                                                              - - ACC TAC CCG CCC AAA GAT AGT ATT TTT CGA GG - #A GAA TTC AAT AGT        TTC      992                                                                    Thr Tyr Pro Pro Lys Asp Ser Ile Phe Arg Gl - #y Glu Phe Asn Ser Phe                          175  - #               180  - #               185              - - ATT AAT CCC ATA ATC CAA TTT CTA GTA CAA CA - #T AAC CTT CCA CTC TTA         1040                                                                       Ile Asn Pro Ile Ile Gln Phe Leu Val Gln Hi - #s Asn Leu Pro Leu Leu                       190      - #           195      - #           200                  - - GCC AAT GTC TAT CCT TAT TTT GGT CAC ATT TT - #C AAC ACT GCT GAT GTC         1088                                                                       Ala Asn Val Tyr Pro Tyr Phe Gly His Ile Ph - #e Asn Thr Ala Asp Val                   205          - #       210          - #       215                      - - CCA CTT TCT TAT GCT TTG TTC ACA CAA CAA GA - #A GCA AAT CCT GCA GGA         1136                                                                       Pro Leu Ser Tyr Ala Leu Phe Thr Gln Gln Gl - #u Ala Asn Pro Ala Gly               220              - #   225              - #   230                          - - TAT CAA AAT CTT TTT GAT GCC CTT TTG GAT TC - #T ATG TAT TTT GCT GTA         1184                                                                       Tyr Gln Asn Leu Phe Asp Ala Leu Leu Asp Se - #r Met Tyr Phe Ala Val           235                 2 - #40                 2 - #45                 2 -      #50                                                                              - - GAG AAA GCT GGA GGA CAA AAT GTG GAG ATT AT - #T GTA TCT GAA AGT        GGC     1232                                                                    Glu Lys Ala Gly Gly Gln Asn Val Glu Ile Il - #e Val Ser Glu Ser Gly                          255  - #               260  - #               265              - - TGG CCT TCT GAA GGA AAC TCT GCA GCA ACT AT - #T GAA AAT GCT CAA ACT         1280                                                                       Trp Pro Ser Glu Gly Asn Ser Ala Ala Thr Il - #e Glu Asn Ala Gln Thr                       270      - #           275      - #           280                  - - TAC TAT GAA AAT TTG ATT AAT CAT GTG AAA AG - #C GGG GCA GGA ACT CCA         1328                                                                       Tyr Tyr Glu Asn Leu Ile Asn His Val Lys Se - #r Gly Ala Gly Thr Pro                   285          - #       290          - #       295                      - - AAG AAA CCT GGA AAG GCT ATA GAA ACT TAT TT - #A TTT GCC ATG TTT GAT         1376                                                                       Lys Lys Pro Gly Lys Ala Ile Glu Thr Tyr Le - #u Phe Ala Met Phe Asp               300              - #   305              - #   310                          - - GAA AAT AAT AAG GAA GGA GAT ATC ACA GAG AA - #A CAC TTT GGA CTC TTT         1424                                                                       Glu Asn Asn Lys Glu Gly Asp Ile Thr Glu Ly - #s His Phe Gly Leu Phe           315                 3 - #20                 3 - #25                 3 -      #30                                                                              - - TCT CCT GAT CAG AGG GCA AAA TAT CAA CTC AA - #T TTC AAT TAATTAATGC          1473                                                                      Ser Pro Asp Gln Arg Ala Lys Tyr Gln Leu As - #n Phe Asn                                       335  - #               340                                     - - ATGGTAACAT TTATTGATAT ATATAGTGAT ATGAGTAATA AGGAGAAGTA GA -             #ACTGCTAT   1533                                                                 - - GTTTTTCTCT TCAATTGAAA ATGTAACTCT GGTTTCACTT TGATATTTAT AT -            #GACATATT   1593                                                                 - - TATTGAGATC TCGTCTTTTG TTTTAAATTC TTGCCTTCTA TTGGCAAATA TC -            #TGCGTAAT   1653                                                                 - - TTTCATTTGT TTTAAAAATT ACTAAGCCTC AAAAGAGTGA CTACCAATAT AT -            #TCTTGATT   1713                                                                 - - ATTAATATTC CCCGTGCTTG GGGGACCGGG TGAGGTGGGG GGTGGGGGGG AT -            #GACGAAAA   1773                                                                 - - AAGTTAATGA AAAACCGGTT TGCATTGGAT GCTCTTTTTA ACCTCCCCAA AA -            #TATGATGG   1833                                                                 - - TTTTGTTGTC TTGGAGAGTG TTTAAGCTAC TTCTTCTCAA GAATTTTCTT GG -            #TCAGTTCT   1893                                                                 - - TAACGTAATT GCTTTTAATT TCTTAATTAT CGGTAACCCT TCGAAACAAA AG -            #GAAAATTA   1953                                                                 - - AGCTAGGAGA TGACTCGTAT TCATAATGTT TTACCTTGGA TCAACCCCGC CT -            #TTATATTT   2013                                                                 - - CATACGA                 - #                  - #                       - #        2020                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Met Thr Leu Cys Ile Lys Asn Gly Phe Leu Al - #a Ala Ala Leu Val        Leu                                                                               1               5 - #                 10 - #                 15             - - Val Gly Leu Leu Ile Cys Ser Ile Gln Met Il - #e Gly Ala Gln Ser Ile                   20     - #             25     - #             30                  - - Gly Val Cys Tyr Gly Lys His Ala Asn Asn Le - #u Pro Ser Asp Gln Asp               35         - #         40         - #         45                      - - Val Ile Asn Leu Tyr Asn Ala Asn Gly Ile Ar - #g Lys Met Arg Ile Tyr           50             - #     55             - #     60                          - - Asn Pro Asp Thr Asn Val Phe Asn Ala Leu Ar - #g Gly Ser Asn Ile Glu       65                 - # 70                 - # 75                 - # 80       - - Ile Ile Leu Asp Val Pro Leu Gln Asp Leu Gl - #n Ser Leu Thr Asp Pro                       85 - #                 90 - #                 95              - - Ser Arg Ala Asn Gly Trp Val Gln Asp Asn Il - #e Ile Asn His Phe Pro                  100      - #           105      - #           110                  - - Asp Val Lys Phe Lys Tyr Ile Ala Val Gly As - #n Glu Val Ser Pro Gly              115          - #       120          - #       125                      - - Asn Asn Gly Gln Tyr Ala Pro Phe Val Ala Pr - #o Ala Met Gln Asn Val          130              - #   135              - #   140                          - - Tyr Asn Ala Leu Ala Ala Ala Gly Leu Gln As - #p Gln Ile Lys Val Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Ala Thr Tyr Ser Gly Ile Leu Ala Asn Th - #r Tyr Pro Pro Lys        Asp                                                                                             165  - #               170  - #               175             - - Ser Ile Phe Arg Gly Glu Phe Asn Ser Phe Il - #e Asn Pro Ile Ile Gln                  180      - #           185      - #           190                  - - Phe Leu Val Gln His Asn Leu Pro Leu Leu Al - #a Asn Val Tyr Pro Tyr              195          - #       200          - #       205                      - - Phe Gly His Ile Phe Asn Thr Ala Asp Val Pr - #o Leu Ser Tyr Ala Leu          210              - #   215              - #   220                          - - Phe Thr Gln Gln Glu Ala Asn Pro Ala Gly Ty - #r Gln Asn Leu Phe Asp      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Ala Leu Leu Asp Ser Met Tyr Phe Ala Val Gl - #u Lys Ala Gly Gly        Gln                                                                                             245  - #               250  - #               255             - - Asn Val Glu Ile Ile Val Ser Glu Ser Gly Tr - #p Pro Ser Glu Gly Asn                  260      - #           265      - #           270                  - - Ser Ala Ala Thr Ile Glu Asn Ala Gln Thr Ty - #r Tyr Glu Asn Leu Ile              275          - #       280          - #       285                      - - Asn His Val Lys Ser Gly Ala Gly Thr Pro Ly - #s Lys Pro Gly Lys Ala          290              - #   295              - #   300                          - - Ile Glu Thr Tyr Leu Phe Ala Met Phe Asp Gl - #u Asn Asn Lys Glu Gly      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asp Ile Thr Glu Lys His Phe Gly Leu Phe Se - #r Pro Asp Gln Arg        Ala                                                                                             325  - #               330  - #               335             - - Lys Tyr Gln Leu Asn Phe Asn                                                          340                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2509 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: join(85..142 - #, 930..1948)                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - AATATAAATA GCTCGTTGTT CATCTTAATT CTCCCAACAA GTCTTCCCAT CA -             #TGTCTACC     60                                                                 - - TCACATAAAC ATAATACTCC TCAA ATG GCT GCT ATC ACA C - #TC CTA GGA TTA           111                                                                                        - #         Met Ala Ala Ile Thr - #Leu Leu Gly Leu                            - #           1       - #        5                           - - CTA CTT GTT GCC AGC AGC ATT GAC ATA GCA  - #G GTTTCTGGTC AAATATTTGA         162                                                                        Leu Leu Val Ala Ser Ser Ile Asp Ile Ala                                        10                 - # 15                                                     - - ACTTCCCAGC CAAAAATATT GTCTTATAAT TTTGTGTGCG CAAAATTTTA AT -             #TTAGTTGA    222                                                                 - - TAGTTATTTG CTTATTTTTC TTTTCAAATT GCTTGTGTTT TTTTCTCAAA TT -            #AACTTGCA    282                                                                 - - CCGTATTCAT TTAGCGATAG TTATTTGCTC TATTTTGTGT AACACTCACT CA -            #CAAACTTT    342                                                                 - - TCAATTTGAG GGGAGGACAG TGAATCTAAG ATTGAAATTT ATGAGTTTAA TT -            #AGACTAAT    402                                                                 - - TCCCATTTGA TTTATTGGCT AGAAGTCAAT TATTTGCATA GTGAGTCTTT TA -            #ACACACAG    462                                                                 - - ATTTGAGTTA AAGCTACTAC GTTCGTATTA ACCCATAACA TATACACCTT CT -            #GTTCTAAT    522                                                                 - - TTCTTTGACA CTTTTTGTTA GTTTGTTCCA AAAAGGACGG ACATATTTGA TA -            #TTTGAGAA    582                                                                 - - TACTTTACCT TAACCTTAAT AGAATTTTTT ATGACATCAC ATATATTATG GA -            #ATATATAC    642                                                                 - - GACCATAATT TTCAAATATC TTATAGTCGT ACAAATATTA TAGCATGTTT AA -            #TACCACAA    702                                                                 - - CTTTCAAATT CTTCTTTTCC TTAAAAACAA AATATGTCAC ATAAATTAAA AT -            #AGAGGAAG    762                                                                 - - TATACTACAT CAATCAGCCC CTAGTGGAGG GGACCTACTG TAAGTTTTTA AG -            #TTTTCAAG    822                                                                 - - AATTCAGTAA TTGATTAGGA GCCCGTCTGG ACATAAAAAA AAATTCCTTT TT -            #TTCCAAAA    882                                                                 - - AATGCCCACT AAATTTCTAA CACTATTTTG TAATTCTTAT TGAGCAG  G - #G GCT        CAA      937                                                                                      - #                  - #                Gly - #Ala        Gln                                                                                               - #                  - #                 20                  - - TCG ATA GGT GTT TGC TAT GGA ATG CTA GGC AA - #C AAC TTG CCA AAT        CAT      985                                                                    Ser Ile Gly Val Cys Tyr Gly Met Leu Gly As - #n Asn Leu Pro Asn His                   25         - #         30         - #         35                      - - TGG GAA GTT ATA CAG CTC TAC AAG TCA AGA AA - #C ATA GGA AGA CTG AGG         1033                                                                       Trp Glu Val Ile Gln Leu Tyr Lys Ser Arg As - #n Ile Gly Arg Leu Arg                40             - #     45             - #     50                          - - CTT TAT GAT CCA AAT CAT GGA GCT TTA CAA GC - #A TTA AAA GGC TCA AAT         1081                                                                       Leu Tyr Asp Pro Asn His Gly Ala Leu Gln Al - #a Leu Lys Gly Ser Asn            55                 - # 60                 - # 65                 - # 70       - - ATT GAA GTT ATG TTA GGA CTT CCC AAT TCA GA - #T GTG AAG CAC ATT GCT         1129                                                                       Ile Glu Val Met Leu Gly Leu Pro Asn Ser As - #p Val Lys His Ile Ala                            75 - #                 80 - #                 85              - - TCC GGA ATG GAA CAT GCA AGA TGG TGG GTA CA - #G AAA AAT GTT AAA GAT         1177                                                                       Ser Gly Met Glu His Ala Arg Trp Trp Val Gl - #n Lys Asn Val Lys Asp                        90     - #             95     - #            100                  - - TTC TGG CCA GAT GTT AAG ATT AAG TAT ATT GC - #T GTT GGG AAT GAA ATC         1225                                                                       Phe Trp Pro Asp Val Lys Ile Lys Tyr Ile Al - #a Val Gly Asn Glu Ile                   105          - #       110          - #       115                      - - AGC CCT GTC ACT GGC ACA TCT TAC CTA ACC TC - #A TTT CTT ACT CCT GCT         1273                                                                       Ser Pro Val Thr Gly Thr Ser Tyr Leu Thr Se - #r Phe Leu Thr Pro Ala               120              - #   125              - #   130                          - - ATG GTA AAT ATT TAC AAA GCA ATT GGT GAA GC - #T GGT TTG GGA AAC AAC         1321                                                                       Met Val Asn Ile Tyr Lys Ala Ile Gly Glu Al - #a Gly Leu Gly Asn Asn           135                 1 - #40                 1 - #45                 1 -      #50                                                                              - - ATC AAG GTC TCA ACT TCT GTA GAC ATG ACC TT - #G ATT GGA AAC TCT        TAT     1369                                                                    Ile Lys Val Ser Thr Ser Val Asp Met Thr Le - #u Ile Gly Asn Ser Tyr                          155  - #               160  - #               165              - - CCA CCA TCA CAG GGT TCG TTT AGG AAC GAT GC - #T AGG TGG TTT GTT GAT         1417                                                                       Pro Pro Ser Gln Gly Ser Phe Arg Asn Asp Al - #a Arg Trp Phe Val Asp                       170      - #           175      - #           180                  - - GCC ATT GTT GGC TTC TTA AGG GAC ACA CGT GC - #A CCT TTA CTC GTT AAC         1465                                                                       Ala Ile Val Gly Phe Leu Arg Asp Thr Arg Al - #a Pro Leu Leu Val Asn                   185          - #       190          - #       195                      - - ATT TAC CCC TAT TTC AGT TAT TCT GGT AAT CC - #A GGC CAG ATT TCT CTC         1513                                                                       Ile Tyr Pro Tyr Phe Ser Tyr Ser Gly Asn Pr - #o Gly Gln Ile Ser Leu               200              - #   205              - #   210                          - - CCC TAT TCT CTT TTT ACA GCA CCA AAT GTG GT - #G GTA CAA GAT GGT TCC         1561                                                                       Pro Tyr Ser Leu Phe Thr Ala Pro Asn Val Va - #l Val Gln Asp Gly Ser           215                 2 - #20                 2 - #25                 2 -      #30                                                                              - - CGC CAA TAT AGG AAC TTA TTT GAT GCA ATG CT - #G GAT TCT GTG TAT        GCT     1609                                                                    Arg Gln Tyr Arg Asn Leu Phe Asp Ala Met Le - #u Asp Ser Val Tyr Ala                          235  - #               240  - #               245              - - GCC CTC GAG CGA TCA GGA GGG GCA TCT GTA GG - #G ATT GTT GTG TCC GAG         1657                                                                       Ala Leu Glu Arg Ser Gly Gly Ala Ser Val Gl - #y Ile Val Val Ser Glu                       250      - #           255      - #           260                  - - AGT GGC TGG CCA TCT GCT GGT GCA TTT GGA GC - #C ACA TAT GAC AAT GCA         1705                                                                       Ser Gly Trp Pro Ser Ala Gly Ala Phe Gly Al - #a Thr Tyr Asp Asn Ala                   265          - #       270          - #       275                      - - GCA ACT TAC TTG AGG AAC TTA ATT CAA CAC GC - #T AAA GAG GGT AGC CCA         1753                                                                       Ala Thr Tyr Leu Arg Asn Leu Ile Gln His Al - #a Lys Glu Gly Ser Pro               280              - #   285              - #   290                          - - AGA AAG CCT GGA CCT ATT GAG ACC TAT ATA TT - #T GCC ATG TTT GAT GAG         1801                                                                       Arg Lys Pro Gly Pro Ile Glu Thr Tyr Ile Ph - #e Ala Met Phe Asp Glu           295                 3 - #00                 3 - #05                 3 -      #10                                                                              - - AAC AAC AAG AAC CCT GAA CTG GAG AAA CAT TT - #T GGA TTG TTT TCC        CCC     1849                                                                    Asn Asn Lys Asn Pro Glu Leu Glu Lys His Ph - #e Gly Leu Phe Ser Pro                          315  - #               320  - #               325              - - AAC AAG CAG CCC AAA TAT AAT ATC AAC TTT GG - #G GTC TCT GGT GGA GTT         1897                                                                       Asn Lys Gln Pro Lys Tyr Asn Ile Asn Phe Gl - #y Val Ser Gly Gly Val                       330      - #           335      - #           340                  - - TGG GAC AGT TCA GTT GAA ACT AAT GCT ACT GC - #T TCT CTC GTA AGT GAG         1945                                                                       Trp Asp Ser Ser Val Glu Thr Asn Ala Thr Al - #a Ser Leu Val Ser Glu                   345          - #       350          - #       355                      - - ATG TGAGCTGATG AGACACTTGA AATCTCTTTA CATACGTATT CCTTGGATG - #G              1998                                                                       Met                                                                            - - AAAACCTAGT AAAAACAAGA GAAATTTTTT CTTTATGCAA GATACTAAAT AA -             #CATTGCAT   2058                                                                 - - GTCTCTGTAA GTCCTCATGG ATTGTTATCC AGTGACGATG CAACTCTGAG TG -            #GTTTTAAA   2118                                                                 - - TTCCTTTTCT TTGTGATATT GGTAATTTGG CAAGAAACTT TCTGTAAGTT TG -            #TGAATTTC   2178                                                                 - - ATGCATCATT AATTATACAT CAGTTCCATG TTTGATCAGA TTGGGATTTG GT -            #AACTTCAA   2238                                                                 - - TGTTAGTATT ATAATTAGTG TCTTTATCAT TGACTATCAA TTAATCTTTA TT -            #TGGCAAGG   2298                                                                 - - CTTGATATAT TTGAGTTACT CTTAGGTATT TGCAAGCAAC TGATCTTTCT TT -            #TATCCCGT   2358                                                                 - - TTCTGGCTTA AACCTCATTA GAAATATATT ATAATGTCAC CTACTCTGTG GT -            #TTAAGACA   2418                                                                 - - TTCCCTTACA TTATAAGGTA TTTCACGTCG TATCAGGTCG AAAAAAATAA TG -            #GTACGCTC   2478                                                                 - - TTTCTTATCA CAAATTTCTC TAACTTCTAG A        - #                  - #            2509                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 359 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Met Ala Ala Ile Thr Leu Leu Gly Leu Leu Le - #u Val Ala Ser Ser Ile        1               5 - #                 10 - #                 15              - - Asp Ile Ala Gly Ala Gln Ser Ile Gly Val Cy - #s Tyr Gly Met Leu Gly                   20     - #             25     - #             30                  - - Asn Asn Leu Pro Asn His Trp Glu Val Ile Gl - #n Leu Tyr Lys Ser Arg               35         - #         40         - #         45                      - - Asn Ile Gly Arg Leu Arg Leu Tyr Asp Pro As - #n His Gly Ala Leu Gln           50             - #     55             - #     60                          - - Ala Leu Lys Gly Ser Asn Ile Glu Val Met Le - #u Gly Leu Pro Asn Ser       65                 - # 70                 - # 75                 - # 80       - - Asp Val Lys His Ile Ala Ser Gly Met Glu Hi - #s Ala Arg Trp Trp Val                       85 - #                 90 - #                 95              - - Gln Lys Asn Val Lys Asp Phe Trp Pro Asp Va - #l Lys Ile Lys Tyr Ile                  100      - #           105      - #           110                  - - Ala Val Gly Asn Glu Ile Ser Pro Val Thr Gl - #y Thr Ser Tyr Leu Thr              115          - #       120          - #       125                      - - Ser Phe Leu Thr Pro Ala Met Val Asn Ile Ty - #r Lys Ala Ile Gly Glu          130              - #   135              - #   140                          - - Ala Gly Leu Gly Asn Asn Ile Lys Val Ser Th - #r Ser Val Asp Met Thr      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Ile Gly Asn Ser Tyr Pro Pro Ser Gln Gl - #y Ser Phe Arg Asn        Asp                                                                                             165  - #               170  - #               175             - - Ala Arg Trp Phe Val Asp Ala Ile Val Gly Ph - #e Leu Arg Asp Thr Arg                  180      - #           185      - #           190                  - - Ala Pro Leu Leu Val Asn Ile Tyr Pro Tyr Ph - #e Ser Tyr Ser Gly Asn              195          - #       200          - #       205                      - - Pro Gly Gln Ile Ser Leu Pro Tyr Ser Leu Ph - #e Thr Ala Pro Asn Val          210              - #   215              - #   220                          - - Val Val Gln Asp Gly Ser Arg Gln Tyr Arg As - #n Leu Phe Asp Ala Met      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Leu Asp Ser Val Tyr Ala Ala Leu Glu Arg Se - #r Gly Gly Ala Ser        Val                                                                                             245  - #               250  - #               255             - - Gly Ile Val Val Ser Glu Ser Gly Trp Pro Se - #r Ala Gly Ala Phe Gly                  260      - #           265      - #           270                  - - Ala Thr Tyr Asp Asn Ala Ala Thr Tyr Leu Ar - #g Asn Leu Ile Gln His              275          - #       280          - #       285                      - - Ala Lys Glu Gly Ser Pro Arg Lys Pro Gly Pr - #o Ile Glu Thr Tyr Ile          290              - #   295              - #   300                          - - Phe Ala Met Phe Asp Glu Asn Asn Lys Asn Pr - #o Glu Leu Glu Lys His      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Phe Gly Leu Phe Ser Pro Asn Lys Gln Pro Ly - #s Tyr Asn Ile Asn        Phe                                                                                             325  - #               330  - #               335             - - Gly Val Ser Gly Gly Val Trp Asp Ser Ser Va - #l Glu Thr Asn Ala Thr                  340      - #           345      - #           350                  - - Ala Ser Leu Val Ser Glu Met                                                      355                                                                    - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - GGAATTCTGG TACCTCCCGG GAGGATCCAT CTAGAGCTCG AGTAAGCTTC  - #                  50                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 50 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - GGAATTCTGG TACCTCCCGG GAGGATCCAT CTAGAGCTCG AGTAAGCTTC  - #                  50                                                                       __________________________________________________________________________

We claim:
 1. A recombinant polynucleotide comprising: a promoter that isfunctional in plants; a first gene encoding an intracellular β-1,3-glucanase, under the control of said promoter; a terminator, operablylinked to said first gene; and a second gene encoding a selectable orscreenable trait, operably linked to regulatory sequences for properexpression; wherein said gene encoding said intracellularβ-1,3-glucanase is modified in the coding region at the 3'-end of thegene such that not all of the C-terminal amino acids involved invacuolar targeting are present in the expressed protein.
 2. Arecombinant polynucleotide according to claim 1, wherein between about 3and 25 C-terminal amino acids of the intracellular β-1,3-glucanase aredeleted.
 3. A cloning or transformation vector comprising therecombinant polynucleotide of claim
 1. 4. The recombinant polynucleotideof claim 1, wherein a translation stopcodon is inserted in the codingregion at the 3'-end of the gene.
 5. A recombinant polynucleotidecomprising:a promoter that is functional in plants; an open readingframe encoding a vacuolar β-1,3-glucanase, under control of saidpromoter wherein said open reading frame encoding said β-1,3-glucanaseis modified to target said vacuolar β-1,3-glucanase to the apoplast bycreating a translation stopcodon in the open reading frame at the 3'-endresulting in deletion of one or more of the C-terminal amino acids ofsaid vacuolar β-1,3-glucanase necessary for vacuolar targeting; and aterminator operably linked to said open reading frame.
 6. A vectorcomprising the recombinant polynucleotide of claim
 5. 7. AnAgrobacterium strain comprising the vector of claim 6.